RXRα agonist bexarotene inhibits atherosclerosis in ApoE-/- mice by regulating TGF-β1/Smad2 signaling pathway

AIM To observe the effect of retinoid X receptor α （RXRα） agonist bexarotene （Bex） on the proliferation of transforming growth factor β1 （TGF-β1）-induced vascular smooth muscle cells （VSMCs） and atherosclerosis in apolipoprotein E knockout （ApoE^-/-） mice, and to explore the underlying mechanism. METHODS Ten C57BL/6 mice were selected as normal control group, and 30 ApoE^-/- mice were randomly divided into 3 groups： ApoE^-/- group, ApoE^-/-+Bex5 （5 mg·kg^-1·d^-1 Bex） group and ApoE^-/-+Bex10 （10 mg·kg^-1·d^-1 Bex） group. Bex was intragastrically given once a day for 8 weeks. The levels of triglyceride （TG） and total cholesterol （TC） were determined by oxidase method, and select masking method was used to determine serum levels of low-density lipoprotein cholesterol （LDL-C） and high-density lipoprotein cholesterol （HDL-C）. The protein levels of TGF-β1, p-Smad2 and Smad2 were determined by Western blot. HE staining was used to observe the intima of the thoracic aorta. The VSMCs were cultured with tissue patch method, and the proliferation of VSMCs was measured by BrdU incorporation method. RESULTS The serum levels of TG, TC and LDL-C, and the expression of TGF-β1 and p-Smad2 in thoracic aorta in ApoE^-/- group were significantly higher than those in C57BL/6 group （P<0.01）. Bex increased p-Smad2 protein level in thoracic aorta in a dose-dependent manner, inhibited the intimal plaque formation and vascular medial proliferation, and decreased the plaque area in ApoE^-/- mice （P<0.01）. No significant difference in serum levels of TG, TC, HDL-C and LDL-C, and TGF-β1 and Smad2 expression in thoracic aorta among ApoE^-/- group, ApoE^-/-+Bex5 group and ApoE^-/-+Bex10 group was observed. TGF-β1 （0.1~10 μg/L） promoted the proliferation of VSMCs, while Bex （10^-9~10^-7 mol/L） inhibited TGF-β1 （5 μg/L）-induced proliferation of VSMCs in a concentration-dependent manner. Bex （10^-7 mol/L） synergistically promoted the protein level of p-Smad2 in VSMCs induced by TGF-β1 （P<0.01）, but inhibited TGF-β1-induced nuclear translocation of p-Smad2. CONCLUSION RXRα agonist Bex inhibits the formation of atherosclerosis in ApoE^-/- mice, and its mechanism may be related to the regulation of TGF-β1/Smad2 pathway.     

Effect of HOCs implantation on protein expression of TGF-β/Smad signaling pathway in liver tissue of hepatic fibrosis rats

AIM: To investigate the effect of hepatic oval cells (HOCs) on the protein expression of TGF-β/Smad signaling pathway in the liver tissue of hepatic fibrosis rats.METHODS: The SD rat models of liver fibrosis were made by treating with carbon tetrachloride and combined factors. The HOCs was isolated from the model rats. HOCs suspension (0.5 mL at a density of 1×1012cells/L) were transplanted via portal vein into the hepatic fibrosis rats at 8th week and observed continuously for 30 days. Meanwhile, WuLing capsules were used for positive control. The blood samples were collected through trail vein at 8th day, 15th day, 23th day and 30th day after transplantation of HOCs. The levels of aspartate aminotransferase (AST) and alamine aminotransferase (ALT) in serum were determined by enzyme method. The morphological changes of hepatic tissues were observed under microscope with HE and Musson staining. The protein levels of collagen type I (Col-Ⅰ), extracellular-signal regulated protein kinase (ERK), phosphory-lation extracellular regulatedprotein kinases (p-ERK), TGF-β receptor type Ⅰ (TβRⅠ), TGF-β receptor type Ⅱ (TβRⅡ), mothers against decapentaplegic homolog 2/3 (Smad 2/3) and mothers against decapentaplegic homolog (Smad 7) were assessed in liver tissues by Western blotting. RESULTS: In HOCs and WuLing capsules treated groups, the levels of ALT and AST decreased significantly at 15th day, 23th day and 30th day after the transplantation of HOC. The damage degree of hepatic fiber hyperplasia of the liver histological structure reduced notably. The expression levels of Col-Ⅰ, ERK, p-ERK, TβRⅠ and TβRⅡ in liver tissues of hepatic fibrosis rats were down-regulated obviously while the expression of Smad 7 increased significantly.CONCLUSION: The implantation of HOCs prevents the progress of liver fibrosis in rats. The mechanism of action is to inhibit the protein expression of p-ERK, TβRⅠ, TβR Ⅱ for TGF-β/Smad signaling pathway of liver tissue.     

Effect of chronic administration of L-thyroxine on Ca2+/calmodulin-dependent protein kinaseⅡ in rat myocardium

AIM: To investigate the effect of chronic injection of L-thyroxine on Ca²⁺/calmodulin-dependent protein kinaseⅡ (CaMKII) and to explore whether CaMKII directly mediates hyperthyroidism-induced cardiac hypertrophy. METHODS: Twenty male Sprague-Dawley rats were randomly divided into hyperthyroid group and control group with 10 animals each. The animal model was produced by intraperitoneal injection of L-thyroxine (0.2 mg·kg^-1·d^-1) for 3 months. The control animals only received saline vehicle in the same procedures. Heart weight (HW), heart-to-body weight ratio (HW/BW), left ventricular-to-body weight ratio (LVW/BW) and diameter of cardiac myocytes were measured to evaluate cardiac hypertrophy. The ratio of perivascular collagen area to vascular luminal area (PVCA/VA) was used to represent myocrdial fibrosis. Moreover, the mRNA expression of CaMKII and the protein level of CaMKII were measured by real-time RT-PCR and Western blotting, respectively. RESULTS: Intraperitoneal injection of L-thyroxine for 3 months significantly increased HW/BW, LVW/BW, PVCA/VA and diameter of cardiac myocytes by 1.87, 1.84, 1.94 and 2.15 folds, respectively (P<0.05 or P<0.01) as compared with control group. The results of real-time RT-PCR revealed that L-thyroxine injection caused a 60% reduction in the mRNA level of cardiac CaMKII (P<0.05). Furthermore, the results of Western blotting confirmed that the protein expression level of cardiac CaMKII in L-thyroxine group diminished by 21% (P<0.05), but accompanied by a 1.58-fold enhancement of phosphorylated activity of CaMKII (P<0.05). CONCLUSION: Thyroxine decreases the expression level of cardiac CaMKII and increases the activity of CaMKII in the chronic hyperthyroid-induced hypertrophic heart, suggesting that CaMKII participates in the formation and maintenance of cardiac hypertrophy induced by hyperthyroidism in a balanced way.     

Protective effect of adiponectin on glucose and lipid metabolism by suppressing MHCⅡ expression

AIM: To investigate whether the protective effect of adiponectin on glucose and lipid metabolism is achieved through down-regulating major histocompatibility complex class Ⅱ (MHCⅡ) in the adipose tissue. METHODS: Adiponectin knockout (KO) mice and C57BL/6(WT) mice were fed with high-fat diet and standard diet for 24 weeks, respectively. The body weight, fasting blood glucose (FBG), fasting insulin (FINS), homeostasis model assessment of insulin resistance (HOMA-IR), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), hepatic histology, and class Ⅱ trans-activator (CⅡTA), histocompatibility 2 class Ⅱ antigen E beta (H2-Eb1) and cluster of differentiation 74(CD74) mRNA and MHC Ⅱ protein levels in adipose tissue were measured at sacrifice. siRNA targeting MHC Ⅱ and overexpression vector was used in 3T3-L1 cells to explore the effect of adiponectin on the protein level of MHCⅡ. RESULTS: The levels of body weight, FBG, FINS, HOMA-IR, TC, TG, LDL-C, hepatic steatosis, CⅡTA, H2-Eb1 and CD74 mRNA expression, and MHCⅡ protein expression in the KO mice were higher than those in the WT mice that fed with high-fat diet or standard diet. In 3T3-L1 cells, inhibition of adiponectin reversed MHC Ⅱ protein level induced by specific siRNA. The expression of MHC Ⅱ in adipocytes decreased after adiponectin was overexpressed. CONCLUSION: Adiponectin improves glucose and lipid metabolism through suppressing the expression of MHCⅡ in the adipose tissue.     

Effect of compound of Epimedium, Astragalus and Radix Puerariae on expression of ADAM10 in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin expression knock-down

AIM To explore the effect of compound of Epimedium, Astragalus and Radix Puerariae on the expression of a disintegrin and metalloproteinase 10 （ADAM10） in Aβ-induced hippocampal neuron HT22 cells with or without hepcidin （HAMP） expression knock-down for analyzing the pathogenesis of Alzheimer disease （AD） at cell level. METHODS Hippocampal neuron HT22 cells were cultured in vitro and randomly divided into 7 groups： control group, Aβ group （Aβ_25-35-induced HT22 cells）, RNAi group （HAMP gene was silenced in HT22 cells）, Aβ+RNAi group （HAMP gene expression in Aβ_25-35-induced HT22 cells was silenced）, Aβ+TCM group （Aβ_25-35-induced HT22 cells were treated with Epimedium, Astragalus root and Radix Puerariae effective components）, RNAi+TCM group （HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components） and Aβ+RNAi+TCM group （Aβ_25-35-induced HT22 cells with HAMP gene silence were treated with Epimedium, Astragalus root and Radix Puerariae effective components）. The silence efficiency of HAMP siRNA was detected by qPCR and Western blot. The ADAM10 expression in each group was determined by immunofluorescence, qPCR and Western blot. RESULTS The HAMP siRNA-3 sequence had the highest interference efficiency. Compared with control group, the expression levels of ADAM10 in Aβ group, RNAi group and Aβ+RNAi group were decreased （P<0.05）. Compared with Aβ group,the expression levels of ADAM10 in Aβ+RNAi group was also decreased （P<0.05）, and the expression levels of ADAM10 in Aβ+TCM group was increased （P<0.05）. Compared with RNAi group, the expression levels of ADAM10 in Aβ+RNAi group was decreased （P<0.05）, while the expression levels of ADAM10 in RNAi+TCM group was increased （P<0.05）. Compared with Aβ+RNAi group, the expression levels of ADAM10 in Aβ+RNAi+TCM group was increased （P<0.05）. CONCLUSION The effective components of Epimedium, Astragalus and Radix Puerariae compound promotes the expression of ADAM10 in Aβ_25-35-induced HT22 cells, which mechanism may be related to the expression of HAMP.     

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.     

Effect of Ca2+/calmodulin-dependent protein kinaseⅡinhibitor KN-93 on late sodium current in rabbits model of heart failure

AIM: To investigate the change of late sodium current (I_NaL) and the effect of Ca²⁺/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) inhibitor KN-93 on I_NaL in the cardiomyocytes after isoproterenol-induced heart fai-lure (HF) in rabbits. METHODS: The rabbit model of HF was induced by injecting isoproterenol (300 μg·kg^-1·d^-1) for 15 d. One month later, all rabbits received by echocardiography and HE staining to observe the morphological changes of myocardium for evaluating the HF model. The protein expression of Na_V1.5, CaMKⅡδ and phosphorylated CaMKⅡδ was determined by Western blot. The ventricular myocytes were isolated from the rabbits of normal saline (NS) group and HF group by Langendorff perfusion, and the whole-cell patch-clamp technique was used to record I_NaL. RESULTS: Compared with NS group, the heart rate in HF group was increased (P<0.01), the ventricular cavity was enlarged (P<0.05), and the cardiac function was decreased (P<0.01). Compared with NS group, the cardiomyocytes in HF group arranged in disorder, vacuolar degeneration and myocardial interstitial edema were observed, and fibrous tissue increased. The protein levels of Na_V1.5, CaMKⅡδ and phosphorylated CaMKⅡδ in HF group were higher than those in NS group (P<0.01). I_NaL in HF group significantly increased compared with NS group (P<0.01). After adding sea anemone toxin Ⅱ (ATXⅡ), the density of I_NaL in HF group and NS group was significantly increased, but that in HF group increased more obviously than that in NS group (P<0.01). After ATXⅡ had induced stable current, we added KN-93 into NS group and HF group, and we found that the ATXⅡ-increased I_NaL in NS group and HF group was significantly decreased (P<0.05).CONCLUSION: CaMKⅡ inhibitor KN-93 inhibits the increase in I_NaL in HF rabbits, which may be related to the activity of CaMKⅡδ and the regulation of CaMKⅡ δ on I_NaL.     

Effect of pioglitazone on glucose metabolism and PPAR-γ expression in lipid-infused rats

AIM: To investigate the effect of pioglitazone (Pio) on glucose metabolism and peroxisome proliferators-activated receptor (PPAR)-γ expression in free fatty acid (FFA) -induced insulin resistance in rats. METHODS: A hyperinsulinaemic-euglycaemic clamp and ［3-3H］-glucose tracing technique were used in awake rats. Glucose metabolism in vivo and PPAR-γ in adipose tissue expression were assessed with elevation FFA by lipid infusion over 4 h in rats pretreated with or without Pio.RESULTS: During steady-state of clamp, there was a significant increase in plasma FFA in two lipid-infused groups, compared to control rats (P<0.01). The glucose infusion rates (GIR) in Pio-treated rats (P/L group), compared with controls, were significantly reduced ［(20.6±0.4) mg·kg^-1·min^-1 vs (33.6±0.6)mg·kg^-1· min^-1, P<0.01］, whereas the GIR was lower in the lipid group (L group) than that in the P/L group［(12.6±0.8) mg·kg^-1·min^-1 vs (20.6±0.4) mg·kg^-1·min^-1, P<0.01］. The hepatic glucose production (HGP) was significantly suppressed (85%) ［(18.3±2.1)mg· kg^-1·min^-1 (basal) vs (2.7±2.4)mg· kg^-1·min^-1, and (17.5±2.6) mg· kg^-1·min^-1 vs (2.6±1.0)mg· kg^-1·min^-1］, all P<0.01 during clamp in control and P/L groups. The suppressive effect of insulin on HGP was significantly blunted in L group［(17.3±2.1)mg· kg^-1·min^-1 vs (15.8±1.5)mg· kg^-1·min^-1］. The rate of glucose disappearance (GRd) was significantly reduced in two lipid-infused rats compared with controls［(26.6±1.6)mg· kg^-1·min^-1 and (23.2±0.9)mg· kg^-1·min^-1 vs (37.7±2.6)mg·kg^-1·min^-1,P<0.01］. The PPAR-γ expression of adipose tissue in P/L group was significantly upregulated. CONCLUSION: Lipid-infusion induces an acute insulin-resistance in vivo. Pio treatment upregulates the PPAR-γ of adipose tissue and suppresses HGP. Pio can protect partly against lipid-induced insulin resistance.     

Effect of PPARδ activation on expression of MMP-9 and fibronectin in infarcted myocardium

AIM:To investigate the effect of peroxisome proliferator-activated receptor δ (PPARδ) activation with dietary GW610742X on the expression of matrix metalloproteinase-9 (MMP-9) and fibronectin (FN) in infarcted and remodeling myocardium. METHODS: Wistar rats were divided into 4 groups: control group, sham group, myocardial infarction (MI) group and MI+GW610742X (GW) group. The left coronary artery was ligated to establish the MI model. PPARδ activator GW610742X (100 mg·kg^-1·d^-1) was given to the rats in GW group. At the 3rd month of the procedure, the expression of PPARδ, MMP-9 and FN at mRNA and protein levels in the left ventricular free wall(LVFW) of the heart from each group was identified and the distribution of FN was detected by immunofluorescence. RESULTS: After 3 months following the procedure, obvious necrosis and fibrosis in LVFW were observed in MI group. The expression of PPARδ in MI group was higher than that in control, sham and GW groups (P<0.01), and PPARδ expression in GW group was lower than that in control and sham group (P<0.05). In MI and GW groups, the expression of MMP-9 was higher while the expression of FN was lower than those in control and sham group (P<0.05 or P<0.01). In GW group, the expression of MMP-9 was lower (P<0.05) while the expression of FN was higher (P<0.01) than those in MI group. Meanwhile, the expression of MMP-9 and FN in sham group was similar to those in control group (P>0.05). CONCLUSION: MMP-9 is upregulated and FN is downregulated in infarcted myocardium during the remodeling process. Activation of PPARδ inhibits the upregulation of MMP-9 and degradation of FN, thus ameliorating the myocardial remodeling.     

Effect of inhibiting nuclear factor-kappa B on TNF-α-induced expression of angiotensinogen and production of angiotensinⅡ in glomerular mesangial cells

AIM: To study the effect of inhibiting nuclear factor-kappa B (NF-κB) activity on the expression of angiotensinogen (AGT) and the production of angiotensinⅡ (AngⅡ) induced by tumor necrosis factor-α (TNF-α) in glomerular mesangial cells (MCs) of SD rats. METHODS: The MCs of SD rats were isolated and divided into three groups as follows: control; MCs treated with TNF-α, and the MCs treated with TNF-α + pyrrolidinedithiocarbamate (PDTC). The activity of nuclear factor-kappa B was measured by electrophoretic mobility shift assay. The expression of AGT was determined by RT-PCR for mRNA and Western blotting for protein. The concentration of angiotensinⅡ in supernatant was measured by RIA. RESULTS: The NF-κB activity in the MCs treated with TNF-α (20.67±9.14)×102 μg/cell was significantly higher than that in control cells ［(8.25±4.35)×102 μg/cell, P<0.01］ and the MCs treated with TNF-α+PDTC ［(7.20±4.57)×102 μg/cell, P<0.01］, and no significant difference was found between control and the MCs treated with TNF-α＋PDTC (P>0.05). The AGT mRNA level in the MCs treated with TNF-α (0.27±0.05) was higher than that in the control cells (0.20±0.05, P<0.05), and no significant difference was observed when compared with that in the MCs treated with TNF-α＋PDTC (0.22±0.06, P>0.05). The expression of AGT protein in the MCs treated with TNF-α (0.60±0.19) μg/cell was higher than that in the control ［(0.37±0.15)μg/cell, P＜0.05］ and the MCs treated with TNF-α＋PDTC ［(0.37±0.17)μg/cell, P<0.05］, and no significance was found between the MCs treated TNF-α＋PDTC and the control (P>0.05). The AngⅡ level in supernatant of cultured MCs treated with TNF-α ［(9.73±2.38)×10-5 ng·L-1/cell］ was significantly higher than that in the control ［(7.50±1.51)×10-5 ng·L-1/cell, P<0.05］ and in the MCs treated with TNF-α＋PDTC ［(6.94±1.46)×10-5 ng·L-1/cell, P<0.05］, however, the difference between the MCs treated with TNF-α＋PDTC and the control was of no significance (P>0.05). CONCLUSION: TNF-α activates the NF-κB in glomerular MCs, induces the AGT expression and the production of AngⅡ. Inhibition of NF-κB decreases the AGT expression and the production of AngⅡ. Therefore, the effects of TNF-α on AGT and AngⅡ may be mediated by NF-κB.     

Effect of Notch1/Hes1 signaling pathway on proliferation and differentiation of AECⅡ by regulating expression of C/EBPα

AIM: To investigate whether Notch1/Hes1 signaling pathway regulates the expression of CCAAT/enhancer binding protein alpha (C/EBPα), thus affecting the proliferation and differentiation of type Ⅱ alveolar epithelial cells (AECⅡ). METHODS: Human AECⅡ were cultured in vitro, and randomly divided into control group, activator group (adding Notch pathway activator Jagged1 protein, 500 μg/L) and inhibitor group (adding Notch inhibitor DAPT, 10 μmol/L). AECⅡ in each group were collected after 24 h of intervention. The expression of Notch1, Hes1 and C/EBPα at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell proliferation ability of the AECⅡ was measured by living cell counting and CCK-8 assay. The cell cycle distribution and differentiation of the AECⅡ were analyzed by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly increased in activator group (P<0.05), AECⅡ entered G₂/M phase from S phase, the proliferation of AECⅡ was increased, and the differentiation of AECⅡ was reduced (P<0.05). The mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly reduced in inhibitor group (P<0.05), the cell cycle of AECⅡ cells was arrested in G₀/G₁ phase, the proliferation of AECⅡ cells was reduced, and the differentiation of AECⅡ cells was increased (P<0.05). CONCLUSION: Notch1/Hes1 signaling pathway regulates the expression of C/EBPα and affects the proliferation and differentiation of AECⅡ.     

Role of nucleolin on myocardial hypertrophy and fibrosis in type Ⅱ diabetic cardiomyopathy mice

AIM:To investigate the effect of nucleolin on diabetic cardiomyopathy in mice.METHODS:A type Ⅱ diabetic cardiomyopathy mouse model was prepared using a cardiac-specific nucleolin-overexpressing transgenic mice.The mice were divided into wild-type mouse control group,nucleolin transgenic mouse control group,wild-type mouse diabetes group and nucleolin transgenic mouse diabetes group.Wheat germ agglutinin (WGA) fluorescent dye,Masson staining and PowerLab system detection were used to further clarify the role of nucleolin on cardiac hypertrophy,fibrosis and cardiac function in type Ⅱ diabetic cardiomyopathy mice.RESULTS:Compared with wild-type mouse control group,no significant increase in blood glucose level was found,while genetical myocardial cell hypertrophy was significantly attenuated in nucleolin transgenic mouse diabetes group.The collagen fibers were also significantly reduced,and hemodynamic indexes±dp/dt_max,left ventricular end-diastolic pressure,left ventricular systolic pressure and heart rate were also improved.The above differences were statistically significant.CONCLUSION:Nucleolin may reduce the occurrence of myocardial hypertrophy and fibrosis,thus improving the cardiac function of diabetic cardiomyopathy mice.     

Effect of interfering TGF-β receptor Ⅱ expression on viability,differentiation and apoptosis of NB4 cells

AIM: To evaluate the effect of interfering TGF-β receptor Ⅱ (TβRⅡ) expression on the viability and differentiation of human acute promyelocytic leukemia NB4 cells induced by all-trans retinoic acid (ATRA) and their apoptosis induced by arsenic trioxide (ATO). METHODS: The technique of lentivirus-mediated RNA interference was used to obtain stable NB4 cells with TβRⅡ knockdown, named TβRⅡ-shRNA NB4 cells. CCK-8 assay was used to detect the viability of TβRⅡ-shRNA NB4 cells. The expression level of CD11b was analyzed by flow cytometry, and Wright-Giemsa staining was used to detect the effects of ATRA on the differentiation of TβRⅡ-shRNA NB4 cells. Double staining (Annexin V-FITC/PI) and AO/EB staining were used to detect the effects of ATO on the apoptosis of TβRⅡ-shRNA NB4 cells. RESULTS: The viability of TβRⅡ-shRNA NB4 cells was significantly higher than that of NB4 parental cells. The differentiation was induced in TβRⅡ-shRNA NB4 cells and NB4 parent cells by treatment with ATRA at different concentration (0.01, 0.02, 0.04, 0.08, 0.1 μmol/L) for 96 h. The differentiation rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells in a dose-dependent manner. ATO induced apoptosis of TβRⅡ-shRNA NB4 cells and NB4 parent cells at different concentrations (2, 4 and 8 μmol/L) for 24 h. The apoptotic rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells dose-dependently. At the concentration of 8 μmol/L for 24 h, the apoptotic rates in TβRⅡ-shRNA NB4 cells and NB4 cells were (49.15±2.05)% and (66.85±2.41)%, respectively (P<0.01). CONCLUSION: Down-regulation of TβRⅡ increases the viability of NB4 cells, inhibits NB4 cell differentiation induced by ATRA, and also inhibits apoptosis induced by ATO.     

Effect of Yangjing-Zhongyu decoction on in vitro maturation and expression of PDGFA and PDGFRα of mouse oocytes

AIMTo investigate the effects of Yangjing-Zhongyu decoction on the in vitro maturation and expression of platelet-derived growth factor subunit A (PDGFA) and platelet-derived growth factor receptor α (PDGFRα) of mouse oocytes. METHODSThe SPF female KM mice were given Yangjing-Zhongyu decoction, and the blood was collected to prepare serum. The serum containing Yangjing-Zhongyu decoction was used to culture immature oocyte-granulosa cell complexes. After the in vitro culture, the oocyte maturation rate, fertilization rate and cleavage rate were observed and calculated, and the expression of PDGFA and PDGFRα in the oocytes at protein and mRNA levels was determined by West?ern blot and real-time PCR. RESULTSYangjing-Zhongyu decoction increased the in vitro maturation rate and fertil?ization rate of the oocytes (P<0.05 or P<0.01), and down-regulated the protein and mRNA expression of PDGFA and PDGFRα (P<0.01). CONCLUSION Yangjing-Zhongyu decoction may promote the in vitro maturation of mouse oocytes by down-regulating the expression of PDGFA and PDGFRα.     

Renal expression of ACE/AngⅡ in ACE2 knockout mice after tourniquet shock and its relationship with renal injury

AIM: To investigate the effect of angiotensin-converting enzyme 2 (ACE2) on tourniquet shock (TS)-induced acute renal injury by observing the renal expression of angiotensin-converting enzyme/angiotensin Ⅱ (ACE/AngⅡ) and injury severity in ACE2 knockout (KO) mice. METHODS: The TS animal model was produced by bilateral tourniquet ligation in the inguinal region on both hind legs for 2 h to induce ischemia, and reperfusion was initiated by cutting latex rings for 4 h. Six-month-old male wild-type (WT) and ACE2 KO C57BL/6 mice were selected, and divided into 4 groups (6 mice in each group) including WT group, WT+TS group, KO group and KO+TS group. The expression of ACE and AngⅡ in the renal tissues was determined by Western blotting and ELISA, respectively. The renal content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD), blood urea nitrogen (BUN) and serum creatinine (Cr) were measured by chemical colorimetry. RESULTS: Compared with WT group, the expression of ACE/AngⅡ was obviously increased in WT+TS group, and significant renal oxidative stress injury was also observed. The expression of ACE/AngⅡ was elevated in KO mice, but no significant renal oxidative stress injury was found. Compared with WT+TS group, more highly increased expression of ACE/AngⅡ and more aggravated renal injury were exhibited in KO+TS group. CONCLUSION: Deletion of ACE2 gene exacerbates TS-induced renal oxidative stress injury by increasing local ACE/AngⅡ expression. The agonist targeting to ACE2 may be helpful for prevention and treatment of TS-induced acute renal injury.     

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.     

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.     

Effect of angiotensin Ⅱ on the remodeling of aorta in spontaneously hypertensive rats

AIM: To investigate the effects of a 10-weeks treatment with angiotensin Ⅱ (Ang Ⅱ) subtype I receptor antagonist losartan on vascular remodeling of thoracic aorta in male spontaneously hypertensive rats (SHR). METHODS: SHR were treated from 16 to 26 weeks of age with losartan at 15 mg/kg·d^-1 or 0.75 mg/kg·d^-1. RESULTS: Losartan (15 mg/kg·d^-1) treatment significantly decreased systolic blood pressure compared with the control group, while losartan (0.75 mg/kg·d^-1) had no the effect, losartan(15 mg) prevents the development of aortic hypertrophy by preventing hypertrophy of vascular smooth muscle cells (VSMC). In the losartan 0.75 group, these parameters were not changed. But in the losartan 15 and losartan 0.75 groups, the collagen content of the aortic media decreased significantly. CONCLUSION: It is inferred that the effect of Ang Ⅱ on stimulating VSMC growth of the aorta in SHR is dependent on arterial pressure, while the effect on collagen fibers is through pressure independent mechanism.     

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.