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.     

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 Kang Xianling decoction on TGF-β1-Smad pathway in a unilateral ureteral obstruction rat model

AIM: To study the effect of Kang Xianling decoction,comprised of dahuang,danshen,taoren,niuxi and danggui,on TGF-β₁-Smad pathway in unilateral ureteral obstruction rat model.METHODS: Eighteen male SD rats were divided into 3 groups,sham group,model group and model group treated with Kang Xianling decoction randomly.Renal interstitial fibrosis model was established in rats by unilateral ureteral obstruction (UUO).After treatment for additional 14 d,parameters of hydroxyproline in obstructed kidney from 3 groups were analyzed.Rats were sacrificed and the pathological statuses of their kidneys were checked by HE staining and electron microscopy.Transforming growth factor-β₁ (TGF-β₁) mRNA in kidney tissue was determined by RT-PCR.TGF-β₁ receptor Ⅰ (TβRⅠ),TGF-β₁ receptorⅡ (TβRⅡ),phosphorylated Smad2 and Smad2 protein were determined by Western blotting.RESULTS: Parameters of hydroxyproline in animals of model group were significantly increased than those in sham operation group (P<0.05).The mRNA expression of TGF-β₁ and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2 in kidney tissue of animals in model group were significantly up-regulated.After intervention with Kang Xianling decoction,the above-mentioned up-regulated parameters,except TGF-β₁,were all significantly inhibited.Compared to model group,the pathological changes in renal tissues in treatment group were remarkable improved.CONCLUSION: Kang Xianling decoction inhibits the TGF-β₁-Smad pathway and the protein expression of TβRⅠ,TβRⅡ,phosphorylated Smad2 and Smad2,so as to decrease the level of collagen in obstructed kidney and to alleviate the renal interstitial fibrosis in UUO rats.     

Effect of caveolin-1 and ERK1/2 on 17β-estradiol-induced inhibition of VSMC proliferation

AIM: To investigate the effect of caveolin-1 and phosphorylation of ERK1/2 on 17β-estradiol (E2) induced inhibition of vascular smooth muscle cells (VSMCs). METHODS: The proliferation in cultured VSMCs was determined by using ［3H］-thymidine incorporation. The expressions of caveolin-1, MKP-1 and ERK1/2 phosphorylation were measured by Western blotting. The expression of caveolin-1 mRNA was measured by RT-PCR. RESULTS: Exposed to fetal calf serum (FCS) for 24 h, the increase in proliferation of VSMCs was detected by ［3H］-thymidine incorporation. Pretreatment with various concentrations of E2 for 24 h inhibited VSMC proliferation induced by FCS. The results of Western blotting and RT-PCR showed that pretreated with 17β-estradiol for 24 h reserved the decrease in caveolin-1 induced by FCS. Western blotting results further proved that the expression of MKP-1 was significantly increased and the expression of ERK1/2 phosphorylation was decreased after incubated with 17β-estradiol. CONCLUSION: 17β-estradiol increases caveolin-1 and MKP-1 expressions, and decreases ERK1/2 phosphorylation, leading to the inhibition of VSMC proliferation.     

Effects of platelet-rich plasma on chondrocyte apoptosis and NLRP3/IL-1β signaling pathway in rabbits with osteoarthritis

AIM To explore the effect of platelet-rich plasma (PRP) on rabbit osteoarthritis and its possible mechanism. METHODS The rabbits with knee osteoarthritis were prepared and then divided into model group, sodium hyaluronate (SH) group and PRP group, and another sham operation group was set up, with 6 rabbits in each group. The gross morphological changes of rabbit cartilage were observed. HE staining was used to evaluate the pathomorphological changes of the cartilage. TUNEL staining was used to detect the apoptosis of chondrocytes. The expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/interleukin-1β （IL-1β） signaling pathway-related molecules was observed by immunohistochemical staining, and the protein levels of caspase-3, Bcl-2 and Bax were determined by Western blot. Chondrocytes were isolated and processed according to grouping, and the NLRP3 and IL-1β levels of the cells were measured by ELISA. RESULTS Compared with sham operation group, Pelletier score, Mankin score, chondrocyte apoptotic rate, the positive protein expression rates of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in model group were increased significantly (P<0.05), while the protein expression of Bcl-2 was decreased significantly (P<0.05). Compared with model group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax in SH group and PRP group were decreased significantly (P<0.05), while the protein expression of Bcl-2 was increased significantly (P<0.05). In PRP group, Pelletier score, Mankin score, the apoptotic rate of chondrocytes, the positive protein expression rates of NLRP3, ASC, caspase-1 and IL-1β, and the protein levels of caspase-3 and Bax were lower than those in SH group, while the protein expression of Bcl-2 was higher than that in SH group (P<0.05). Compared with control group, the expression of NL?RP3 and IL-1β in MCC950 (NLRP3 ihibitor) group were significantly reduced (P<0.05), the expression of NLRP3 in eucalyptol (IL-1β inhibitor) group was not significantly changed (P>0.05), and the expression of IL-1β was significantly reduced (P<0.05). CONCLUSION Platelet-rich plasma promotes the repair of cartilage in osteoarthritis rabbits, which has better effect than SH. The mechanism may be related to the inhibition of NLRP3/IL-1β pathway and the reduction of chondrocyte apoptosis.     

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 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.     

Effects of curcumin on actinomycin D/TNF-α-induced injury in PC12 cells and rat hippocampal neurons

AIM: To study the possible mechanism of curcumin on actinomycin D (ActD)/tumor necrosis factor α (TNF-α)-induced injury in PC12 cells and rat hippocampal neurons. METHODS: PC12 cells were divided into control group, TNF-α group, ActD group, curcumin group, ActD/TNF-α group and curcumin+ActD/TNF-α group. The cells were cultured for 24 h. Inverted fluorescence microscopy was used to observe the morphological changes of the cells in each group. Annexin V/PI double staining was applied to analyze the apoptosis of PC12 cells. The level of intracellular Ca²⁺ was detected by Fluo-3 AM staining. Rat hippocampal slices were prepared and divided into the same groups as the PC12 cells. Extracellular microelectrode recording technique was used to observe and calculate the changes of long-term potentiation (LTP) in different groups. RESULTS: Apoptosis of PC12 cells was induced by ActD/TNF-α. Curcumin protected the PC12 cells from ActD/TNF-α-induced apoptosis (P<0.05). ActD/TNF-α increased the intracellular Ca²⁺ concentration. Curcumin significantly reduced ActD/TNF-α-induced apoptosis by decreasing the intracellular Ca²⁺ concentration (P<0.05), inversed the effect of ActD/TNF-α on LTP in hippocampal slices, and improved the synaptic plasticity (P<0.05). CONCLUSION: Curcumin protects ActD/TNF-α-induced neuronal damage by depressing the intracellular Ca²⁺ concentration and maintaining the homostasis of intracellular calcium.     

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 tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE-/- mice

AIM To observe the effect of tanshinone ⅡA on liver lipid deposition and ferroptosis-related protein expression in ApoE^-/- mice. METHODS Thirty-two ApoE^-/- mice were randomly divided into model group, high-dose (60 mg/kg) tanshinone ⅡA group, low-dose (30 mg/kg) tanshinone ⅡA group and simvastatin group, and C57BL/6J mice (n=8) were used as normal control group. The mice in normal control group were given the basic feeding, while the others were given high-fat diet. The mice in tanshinone ⅡA groups and simvastatin group were given corresponding drugs. The mice in normal control group and model group were intraperitoneally injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by automatic biochemistry analyzer. The liver tissues were stained with HE and oil red O. The contents of reactive oxygen species (ROS) and glutathione (GSH) in liver tissues of the mice were measured by commercially available kits. The liver glutathione peroxidase 4 (GPX4) and p53 were detected by immunohistochemical method. The protein and mRNA expression levels of ferroptosis-related factors GPX4, xCT/SLC7A11, p53 and ferritin heavy chain 1 (FTH1) were determined by Wes automatic Western blot quantitative analysis system and RT-qPCR. RESULTS Compared with normal control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.05 or P<0.01), and HDL-C did not change significantly. The fat vacuoles were clearly visible in liver tissue. The content of ROS in liver tissue was increased significantly,and GSH was decreased significantly (P<0.01). The mRNA and protein expression levels of p53 were increased significantly, and GPX4, xCT/SLC7A11 and FTH1 were decreased significantly (P<0.05 or P<0.01). Compared with model group, tanshinone ⅡA significantly decreased the serum levels of TC, TG and LDL-C (P<0.05 or P<0.01), and HDL-C did not change significantly. High-dose and low-dose tanshinoneⅡA also significantly decreased the degree of steatosis, and the size of lipid droplets. The content of ROS in liver tissues was decreased significantly, and GSH was increased significantly (P<0.01). The mRNA and protein expression levels of GPX4, xCT/SLC7A11 and FTH1 were increased significantly, and p53 were decreased significantly (P<0.05 or P<0.01). CONCLUSION Tanshinone ⅡA reduces liver lipid deposition and lipid peroxidation damage in ApoE^-/- mice, which may be related to the intervention of ferroptosis-related proteins in the liver cells.     

Effect of the acupotome therapy and electro-acupuncture on the serum contents of IL-1β, IL-6 and TNF-α in rabbits with knee osteoarthritis

AIM: IL-1β, IL-6 and TNF-α play important roles in emergence of osteoarthritis. This study aims at observing the effect of the acupotome therapy and electro-acupuncture on the cytokines in serum of rabbits with osteoarthritis. METHODS: 52 New Zealand rabbits were divided randomly into the normal group, model group, acupotome group and electro-acupuncture group. Knee osteoarthritis of the model rabbits was made with the straightened immobilization method. The acupotome and electro-acupuncture therapies were applied for three weeks. One week after the treatment the serum was collected, and the changes of IL-1β, IL-6 and TNF-α in serum were detected with RIA. RESULTS: In comparison with the control group, the contents of IL-1β, IL-6 and TNF-α elevated significantly in the knee osteoarthritis model group (P<0.05). Compared to the knee osteoarthritis model group, the contents of the cytokines in acupotome treatment group and electro-acupuncture treatment group decreased significantly (P<0.05). No significant difference between the content of cytokines in the acupotome treatment or electro-acupuncture treatment groups (P<0.05) was observed, also no statistical difference between the acupotome treatment or electro-cupuncture treatment groups and the control group was found. However, the contents of the three cytokines in the acupotome treatment and electro-acupuncture treatment groups were still higher than those in control group. CONCLUSION: The acupotome and electro-acupuncture treatment can decrease the release of IL-1β, IL-6 and TNF-α, indicating that the two therapies play an important role in improvement of the articular cartilage cell injury and function through inhibiting the generation of matrix protease and alleviation of degradation of the cartilage matrix.     

Effects of Huanglian-jiedu decoction on TLR4/NF-κB signaling pathway and goblet cells of mucosa in rats with allergic rhinitis

AIM To investigate the effect of Huanglian-jiedu decoction （HLJD） on goblet cells and Toll-like receptor 4 （TLR4）/nuclear factor （NF）-κB signaling pathway in allergic rhinitis rats. METHODS The rat model of allergic rhinitis was made by ovalbumin. The model rats were divided into model group, low-, medium- and high-dose HLJD groups, and positive control drug group. The rats in low-, medium- and high-dose HLJD groups were given different doses（5, 10 and 20 g/kg, respectively） of crude drug by intragastric administration, the rats in positive control group was given fluticasone propionate nasal spray （50 μg per side）, and the rats in control group and model group were given normal saline, once per day for 10 days. The behaviors were observed and scored after modeling and treatment, the weight of nasal secretion was measured after the treatment. The goblet cells in nasal mucosa were observed by periodic acid-Schiff （PAS） staining. The morphological changes of nasal mucosa were observed by hematoxylin-eosin （HE） staining. The interleukin （IL）-4 and IL-5 levels in nasal mucosa were measured by ELISA. The mRNA levels of mouse calcium-activated chloride channel 3 （mCLCA3） and mucin 5AC （MUC5AC） were detected by RT-qPCR. The protein expression of TLR4 and NF-κB p65 was determined by Western blot. RESULTS After modeling, compared with control group, the behavioral scores in model group, low-, medium- and high-dose HLJD groups and positive control group were increased （P<0.05）. The eosinophils, neutrophils and lymphocytes in nasal mucosa were seriously infiltrated, inflammatory infiltration was obvious, and obvious small vessel dilatation and interstitial edema in model group were observed. With the increase in the dosage of HLJD, the lymphatic infiltration was obviously relieved, but the eosinophil and neutrophil infiltration still existed, and the inflammatory infiltration was relieved. Compared with control group, the behavioral score, nasal secretion, the relative proportion of goblet cells in the mucosa, the IL-4 and IL-5 levels, the mRNA levels of mCLCA3 and MUC5AC, and the protein expression of TLR4 and NF-κB p65 in the mucosa of model group were increased （P<0.05）. Compared with model group, the behavioral score, nasal secretion, the relative proportion of goblet cells in the mucosa, the IL-4 and IL-5 levels, the mRNA levels of mCLCA3 and MUC5AC, and the protein expression of TLR4 in the mucosa of low-, medium- and high-dose HLJD groups and positive control group were increased （P<0.05）, and the protein expression of NF-κB p65 in the mucosa of medium- and high-dose HLJD groups and positive control group was decreased （P<0.05）. CONCLUSION Huanglian-jiedu decoction reduces the relative proportion of goblet cells in the nasal mucosa of rats with allergic rhinitis, which may be achieved by inhibiting TLR4/NF-κB signaling pathway.     

Effects of Coriaria sinica Maxim’s extract on burn wound healing and scar hyperplasia in rats based on ILK signaling pathway

AIM: To explore the mechanism of Coriaria sinica Maxim’s extract (CSME) promoting burn wound healing in the early stage and inhibiting excessive scar hyperplasia in the later stage, based on the signaling pathways, such as transforming growth factor-β₁ (TGF-β₁) regulated by integrin-linked kinase (ILK) and ILK regulated by PI3K/AKT. METHODS: Female SD rats (n=180; 180~200 g) were randomly divided into 6 groups: normal control (NC) group, vaseline (VL) group, silver sulfadiazine (SS) group and low-, medium- and high-dose of CSME (CSME-L, CSME-M and CSME-H) groups, with 30 rats in each group. Except for the rats in NC group, VL, SS, and 3 doses of CSME were applied to the wound surface of the rats in the corresponding groups every day after II° burn model was made on their waist-back in the condition of chloral hydrate anesthesia. To calculate the healing rate (HR), 10 rats in each experimental group were randomly selected to remove their wound skin for observing the pathologic change, detecting the expression of related proteins by Western blot and RT-qPCR, and checking the collagen shrinkage (SK) by fibroblast culture at the 7th, 14th, and 21st days. RESULTS: The expression of ILK, fibronectin (FN), TGF-β₁, α-smooth muscle actin (α-SMA) and integrin-β₁ (ITG-β₁) at protein and mRNA levels in wound skin of CSME groups was stronger than that in VL group and SS group at the 7th day in a dose-dependent manner, but weaker than that in VL group and SS group at the 21st day (P<0.05). Meanwhile, the protein and mRNA expression of collagen type I (Col I) in CSME groups was stronger than that in VL group and SS group from the 7th day to the 14th day, but weaker than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). However, the protein and mRNA expression of collagen type III (Col III) in CSME groups was weaker than that in VL group and SS group from the 7th day to the 14th day, but stronger than that in VL group and SS group at the 21st day in a dose-dependent manner (P<0.05). The SK of fibroblasts in VL group and SS group was increased continuously over time and reached its peak at 96 h. SK in CSME groups was only higher than that in VL group and SS group at 24 h and 48 h in a dose-dependent manner, but lower than that in VL group and SS group at 96 h (P<0.05). CONCLUSION: CSME promotes burn wound healing in the early stages and inhibits the scar hyperplasia in the later stages. The mechanisms may be related to its multicomponents or multiple-targets to intervene in the signaling pathways such as TGF-β₁ regulated by ILK and ILK regulated by PI3K/AKT. It may also be related to the ratio of Col I and Col III expression.     

Effect of TNF-α and Yigu capsule drug-containing sera on osteoblasts apoptosis in coculture system

AIM: To observe the effect of TNF-α and Yigu capsule drug-containing sera on osteoblasts apoptosis in osteoblasts-osteoclasts coculture system.METHODS: (1) Twenty female Sprague-Dawley rats, 10 months old, were randomly assigned into 2 groups, NS and Yigu capsule groups, to prepare blank sera and drug-containing sera. (2) The DNA gel electrophoresis method was used to detect apoptosis of osteoblasts treated with different concentration of TNF-α in order to determine the best dosage in the co-culture system. (3) The cells were divided into four groups, TNF-α group, normal group, TNF-α + blank serum group and TNF-α + drug-containing serum group. DNA gel electrophoresis, acridine orange staining and flow cytometry were used to observe osteoblast apoptosis in these groups. RESULTS: (1) After induced by TNF-α for 72 h at a concentration of 60 μg/L, relatively typical DNA ladder appeared in TNF-α group. (2) Only two DNA brands appeared and most of cells were well-proportioned stained in TNF-α + drug-containing serum group, the rate of osteoblasts apoptosis in TNF-α + drug-containing serum group (9.60%±0.26%) was obviously lower than that in the TNF-α group (26.90%±0.06%) and TNF-α + blank serum group (18.10%±0.06%). CONCLUSION: TNF-α induces osteoblast apoptosis in the co-culture system, and Yigu capsule drug- containing serum prevents osteoblast apoptosis induced by TNF-α.     

Effects of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells

AIM: To observe the expression of hypoxia-inducible factor-1alpha (HIF-1α) and stromal cell-derived factor 1 (SDF-1) in pulmonary artery endothelial cells (PAECs) of SD rats and to investigate the role of HIF-1α/SDF-1 signaling axis on hypoxia-induced migration and adhesion of progenitor cells to PAECs. METHODS: Immunomagnetic beads were used to separate and purify the CD34⁺/CXCR4⁺ progenitor cells derived from the peripheral circulation of SD rats. The expression of HIF-1α and SDF-1 in PAECs exposed to hypoxia (1% O₂, 5% CO₂ and 94% N₂) was detected by immunofluorescence, Western blotting and ELISA. The migration index and adhesion rate were measured in the progenitor cells, which were subjected to the following different treatments: (1) normoxia (21% O₂); (2) hypoxia 12 h; (3) hypoxia 12 h +HIF-1α inhibitor (2ME2); (4) hypoxia 12 h+SDF-1 neutralizing antibody; (5) hypoxia 12 h+2ME2+SDF-1 neutralizing antibody.RESULTS: The expression of HIF-1α and SDF-1 in PAECs was effectively induced by the hypoxic exposure, and both of them reached the peak levels after 12 h of hypoxic treatment (P<0.01), while administration of 2ME2 decreased the hypoxia-induced SDF-1 expression (P<0.05). Treatment of the PAECs with 2ME2 or SDF-1 neutralizing antibody attenuated the migration index and adhesion rate of progenitor cells to the PAECs (P<0.05). CONCLUSION: There is a HIF-1α/SDF-1 signaling axis in hypoxia-exposed PAECs, which may play a crucial role in the migration and adhesion of progenitor cells to PAECs.     

Effects of arsenic trioxide on activities of MMPs and expression of TIMP-1 and TGF-β1 in skin fibroblasts and polymorphonuclear neutrophils

AIM: To observe the effects of arsenic trioxide (As₂O₃) on activities of matrix metalloproteinases (MMPs), expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and transforming growth factor beta1 (TGF-β₁) in human fibroblast (hFb), and to discuss weather As₂O₃ promotes the healing of chronic skin ulcer through regulating collagen metabolism. METHODS: Zymography was used for testing activity of MMP-9 deriving from rat polymorphonuclear neutrophils (PMNs) and activities of MMP-1, MMP-2 secreted by hFb. Immunocytochemical method was used to determine the expressions of TIMP-1 and TGF-β₁. RESULTS: At the concentration of 50 mg/L, As₂O₃ elevated the activity of MMP-9 (P<0.01). At the concentration of 0.8 mg/L, As₂O₃ increased the activities of MMP-1 and MMP-2 (P<0.01, respectively). After hFb was cultured with As₂O₃ for 6 h, 12 h and 18 h, the expressions of TIMP-1 and TGF-β₁ decreased continuously (P<0.01). CONCLUSION: As₂O₃ elevates the activities of MMP-1, MMP -2 and MMP-9, also inhibits the expressions of TIMP-1 and TGF-β₁, suggesting that arsenic preparation may exert positive effect on healing chronic skin ulcer through regulating collagen metabolism.     

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 stage of harvesting and seed treatment on germination,seedling emergence and growth in Corchorus olitorius ‘Oniyaya’

Fruits of Corchorus olitorius L. were harvested at three different developmental stages on the basis of colour (yellow, yellow with brown patches or completely brown). Seed germination (total and rate) and seedling emergence from soil varied with the fruit colour. Steeping the seeds in water at 97°C for 5 s and seed-coat scarification using sandpaper significantly improved seed germination and seedling emergence. Stage of harvesting also affected the shoot lengths and degree of uniformity in shoot lengths of 5-week-old seedlings.