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Objective:To evaluate the anti-adhesion properties of xyloglucan(mXG)hydrogel in the L929 mouse fibroblasts,to establish the animal model of recurrent adhesion in the rats after adhesionlysis,and to investigate the prevention effect of mXG hydrogel in recurrent adhesion and its influence in the expressions of transforming growth factor-β1(TGF-β1)and connective tissue growth factor(CTGF).Methods:After seeding on the surface of mXG gel,the adhesion property of L929 mouse fibroblasts was detected with fluorescence staining.The rat models of recurrent adhesion were established after adhesionlysis.Fourty-eight SD rats were randomly divided into 3 groups respectively(n=16).In adhesion group,1 mL saline was injected into the abdominal wall and cecum of the rats;in commercial membrane group,the 2 cm×3 cm chitosan anti-adhesion membrane was used to cover the wound of abdominal wall of the rats;in mXG hydrogel group,1 mL 4% mXG hydrogel was injected into the abdominal wall and cecal wound of the rats,and abdominal surgery was ended after the complete formation of the hydrogel(3 min).Eight rats were killed in each group 7 and 14 d after the second operation,and the degrees of adhesion were evaluated and the histological changes were observed. Immunohistochemical staining was used to observe the expression levels of CTGF and TGF-β1.Results:A large number of L929 mouse fibroblasts proliferated well and exhibited a spherical morphology in control group;but in mXG hydrogel group,only very little L929 mouse fibroblasts were globular,showing the mXG hydrogel had good resistance to the adhesion of L929 mouse fibroblasts.Blunt separate adhesion surface formed in all of the rats after operation.7 d after the second operation,4-5 score adhesion with fibrous connective tissue hyperplasia formed in adhesion group;moderate adhesion formed in commercial membrane group with more connective tissue;most cecum and abdominal wall began to heal in mXG hydrogel group with less connective tissue.14 d after the second operation,more severe peritoneal adhesions presented in adhesion group with proliferated fiber connetive tissue and collegan;severe adhesions also presented in commercial membrane group;mild adhesion was found in two rats mXG group,the other rats had no adhesion,and the defects were almost completely recovered.On days 7 and 14 after the second operation,the mean adhesion score of the rats in mXG group was significantly lower than those in adhesion group and commercial membrane group(P<0.05).The expression levels of TGF-β1 and CTGF were increased with the increase of peritoneal adhesion scores and collagen deposition;the expression levels of TGF-β1 and CTGF were the highest in adgesion group and the lowest in mXG group.Conclusion:mXG hydrogels have good resistance to fibroblast adhesion,and mXG hydrogel is effective in reducing the formation of recurrent adhesion in the rats after adhesionlysis and can decrease the expression levels of adhesion-related factors TGF-β1 and CTGF.
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Aim To observe the effects of astaxanthin ( ASTX) on the expression of collegeⅠ( ColⅠ) and type Ⅲcollagen ( Col Ⅲ) of cardiac fibroblasts( CFs) which caused by transforming growth factor β1 ( TGF-β1) and to explore its mechanism of action. Methods CFs were induced by TGF-β1 , and then pretreated with different concentrations of ASTX ( 0 , 5 , 10 , 20 , 40, 80, 160 μmol·L-1) for 24 h. MTT assay was used to determine the activity of CFs. The activation of ROS in CFs cells was detected by DCFH-DA kit. Smad3 gene was silenced by siRNA technique, and re-al-time PCR was used to detect the expression of ColⅠ, Col Ⅲ mRNA before and after Smad3 silencing. Western blot was used to detect the expression of ColⅠ, Col Ⅲ and Smad3 protein levels before and after Smad3 silencing. Results ASTX had no obvious cyto-toxicity in the range of 0 ～20 μmol · L-1 , and could significantly reduce ROS production induced by TGF-β1 in CFs (P<0.05). In addition, ASTX significant-ly inhibited the expression of ColⅠand ColⅢmRNA and protein ( P<0.01 ) of TGF-β1-induced CFs in a concentration-dependent manner. Also, ASTX could significantly down-regulate phosphorylation of Smad3 in TGF-β1-induced CFs ( P <0.01 ) . The expression of Col Ⅰ and Col Ⅲ mRNA and protein was also signifi-cantly down-regulated by Smad3 gene silencing ( P <0.01 ) . Conclusions ASTX can effectively inhibit the expression of Col Ⅰ, Col Ⅲ mRNA and protein of TGF-β1-induced CFs, and the possible mechanism may involve the down-regulation of Smad3 phosphoryla-tion.
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Objective:To research the correlation of serum levels of IL-21,TGF-β1,TNF-α and IgA1 in children with allergic purpura and the purpura nephritis.Methods:57 cases with allergic purpura who were treated in our hospital from November 2015 to May 2016 were selected and 29 cases were diagnosed with general allergic purpura,and another 28 cases were diagnosed with purpuric nephritis.30 healthy children were selected as the control group.Then the serum levels of IL-21,TGF-β1,TNF-α,IgAl,immunoglobulin A (IgA),C3 and C4 between the three groups were observed and compared.Results:The serum levels of IL-21,TGF-β1,TNF-α,IgA1 and IgA in the purpuric nephritis group were higher than those of the control group and the general allergic purpura group,and the differences were statistically significant (P＜0.05);The serum levels of IL-21,TGF-β1,TNF-α,IgA1 and IgA in the general allergic purpura group were higher than those of the control group,and the differences were statistically significant (P＜0.05);There was no statistically significant difference about the C3 and C4 in the three groups (P＞0.05).Conclusion:The serum levels of IL-21,TGF-β1,TNF-α and IgA1 may be involved in the development ofhenoch-schonlein purpura and purpura nephritis.
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OBJECTIVE:To study the effects of salvianolic acid B(Sal B)on angiotensin Ⅱ(Ang Ⅱ)-induced cardiac fibro-blast proliferation,secretion of type Ⅲ collagen,protein expressions of matrix metalloproteinase 9 (MMP-9),Smad2/3,Smad7, and explore its mechanism of anti-myocardial fibrosis. METHODS:Cells were divided into blank control group(culture medium) Ang Ⅱ model group,Sal B low-dose,medium-dose,high-dose groups (12.5,25,50 μmol/L). After cultured 1 h by blank or drug-containing culture,except for blank control group,cells in other groups were added 1 μmol/L Ang Ⅱ to induce proliferation. for 24 h. MTT method and hematoxylin-eosin staining method were adopted investigate the effect of Sal B on proliferation. Western blot method was adopted to detect the effects of Sal B on protein expressions of type Ⅲ collagen,MMP-9,Smad2/3,Smad7. RE-SULTS:Compared with blank control group,cells in Ang Ⅱ model group were significantly proliferated,protein expressions of type Ⅲ collagen,MMP-9,Smad2/3 were obviously enhanced,protein expression of Smad7 was obviously weakened,with statisti-cal significances(P<0.05). Compared with Ang Ⅱ model group,the cell proliferation in Sal B groups were inhibited,protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 were weakened,while protein expression of Smad7 was enhanced. Except the pro-tein expression of type Ⅲ collagen in Sal B low-dose and medium-dose groups,the protein expression of Smad2/3 in Sal B high-dose group did not change significantly,other indexes had statistical significances(P<0.05). CONCLUSIONS:The anti-myo-cardial fibrosis effect of Sal B may be associated with inhibiting the proliferation of cardiac fibroblasts,down-regulating protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 and up-regulating protein expression of Smad7.
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OBJECTIVE:To study the effects of salvianolic acid B(Sal B)on angiotensin Ⅱ(Ang Ⅱ)-induced cardiac fibro-blast proliferation,secretion of type Ⅲ collagen,protein expressions of matrix metalloproteinase 9 (MMP-9),Smad2/3,Smad7, and explore its mechanism of anti-myocardial fibrosis. METHODS:Cells were divided into blank control group(culture medium) Ang Ⅱ model group,Sal B low-dose,medium-dose,high-dose groups (12.5,25,50 μmol/L). After cultured 1 h by blank or drug-containing culture,except for blank control group,cells in other groups were added 1 μmol/L Ang Ⅱ to induce proliferation. for 24 h. MTT method and hematoxylin-eosin staining method were adopted investigate the effect of Sal B on proliferation. Western blot method was adopted to detect the effects of Sal B on protein expressions of type Ⅲ collagen,MMP-9,Smad2/3,Smad7. RE-SULTS:Compared with blank control group,cells in Ang Ⅱ model group were significantly proliferated,protein expressions of type Ⅲ collagen,MMP-9,Smad2/3 were obviously enhanced,protein expression of Smad7 was obviously weakened,with statisti-cal significances(P<0.05). Compared with Ang Ⅱ model group,the cell proliferation in Sal B groups were inhibited,protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 were weakened,while protein expression of Smad7 was enhanced. Except the pro-tein expression of type Ⅲ collagen in Sal B low-dose and medium-dose groups,the protein expression of Smad2/3 in Sal B high-dose group did not change significantly,other indexes had statistical significances(P<0.05). CONCLUSIONS:The anti-myo-cardial fibrosis effect of Sal B may be associated with inhibiting the proliferation of cardiac fibroblasts,down-regulating protein ex-pressions of typeⅢcollagen,MMP-9,Smad2/3 and up-regulating protein expression of Smad7.
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Objective To investigate the inhibitory effect of Rhodiola total flavonoids on transforming growth factor (TGF)-β1-induced proliferation of cardiac fibroblasts (CFB) in the rats, and to explore its mechanisms of improving myocardial fibrosis.Methods 5 μg·L-1 TGF-β1 was used to induce the proliferation of CFB to build the cell models of myocardial fibrosis.The cultured CFB were divided into control group,model group,and low, medium,high doses (25,50 and 100 mg· L-1 )of Rhodiola total flavonoids groups.The cells were treated for 48 h,the vitalities of cells were detected by MTT,the levels of collagen proteinⅠ (ColⅠ)and collagen proteinⅢ(Col Ⅲ)in supernatant were measured by ELISA,and the activities of total superoxide dismutase (T-SOD)and glutathione peroxidase (GSH-Px)were detected,the levels of malondialdehyde (MDA)and glutathione (GSH) were also measured.Results The MTT results indicated that compared with control group,the cell vitality in model group was significantly increased (P < 0.01 ).compared with model group,the cell vitalities of CFB in Rhodiola total flavonoids groups were significantly decreased (P < 0.05 ). Compared with control group, the T-SOD activity in model group was decreased, while the MDA level was significantly increased (P <0.05);compared with model group,the T-SOD activities in 50 and 100 mg· L-1 Rhodiola total flavonoids groups were increased (P <0.01);the MDA levels in 25 and 50 mg· L-1 groups were decreased significantly (P < 0.05). Compared with control group,the GSH-Px activity and GSH level in model group were significantly decreased (P <0.01).Compared with model group,the GSH-Px activities and GSH levels in Rhodiola total flavonoids groups were increased (P <0.05).Compared with control group,the ColⅠand Col Ⅲ levels in model group were significantly increased (P <0.01);compared with model group,the ColⅠand Col Ⅲ levels in Rhodiola total flavonoids groups were significantly decreased (P <0.05).Conclusion Rhodiola total flavonoids can inhibit the proliferation of rat CFB.The development of myocardial fibrosis may be inhibited by Rhodiola total flavonoids through anti-oxidative stress pathway.
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Objective To observe the effects of 1,25-dihydroxyvitamin D3 on the expressions of transforming growth factor-β1(TGF-β1), fibronectin(FN),and vascular endothelial growth factor(VEGF) in rats with diabetic nephropathy(DN), and to elucidate the protective mechanism played by 1,25-dihydroxyvitamin D3. Methods DN models were estabolished by injecting streptozotoein ( STZ ) into male SD rats, which were divided into TGF-β1 overexpression group, TGF-β1 overexpression plus vitamin D3 group, TGF-β1 low-expression group, TGF-β1 low-expression plus vitamin D3 group, TGF-β1 normal-expression group, and TGF-β1 normal-expression plus vitamin D3 group. After 1,25-dihydroxyvitamin D3 treatment for 37 days, renal function and blood biochemical parameters were evaluated. The morphology and fibrosis of kidney tissues were observed. The expressions of TGF-β1, FN, and VEGF in kidney cortex were measured by immunohistochemistry, realtime PCR, and Western blotting. Results The levels of cholesterol, triglyceride, creatinine,plasma glucose, HbA1C , and 24 h urinary protein were lower in vitamin D3treated groups than those in corresponding control groups(P<0. 05). The degree of renal fibrosis was raised with the increased level of TGF-β1. Vitamin D3 treatment decreased the fibrosis in diabetic kidney. There were significant differences in the mRNA and protein expressions of TGF-β1 in three control groups(P<0. 05). With the increased levels of TGF-β1, the expressions of FN and VEGF were increased. The expressions of TGF-β1, FN, and VEGF were lowered by vitamin D3compared with the corresponding control groups(P<0. 05). Conclusion 1,25-dihydroxy-vitamin D3 may protect the renal tissure in diabetic rats via inhibiting the expressions of TGF-β1, FN, and VEGF in the kidney.
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Objective To investigate the effects of Ruangan granule on transforming growth factor-β1(TGF-β1)/Smads signaling pathway in liver fibrosis in rats. Methods A total of 105 Wistar rats were randomly divided into normal control group, model group and colchicine, Dahuang-Zhechong pill group, high-, medium- and low-dose Ruangan granule groups (n=15 in each group). Liver fibrosis was induced by carbon tetrachloride and a high-cholesterol diet. After modeling, the low-, medium- and high-dose Ruangan granule groups were intragastric administrated Ruangan granule mixed suspension 3.6, 7.2, 14.4 g/(kg?d), respectively;Dahuang-Zhechong pill group was administrated with Dahuang-Zhechong pellets mixed suspension of 0.18 g/(kg?d);the colchicine group was intragastric administrated with colchicine mixed suspension of 0.108 mg/(kg?d);and the normal control group and the model group were intragastric administrated with the equal volume of distilled water. All rats were intragastric administrated for 8 weeks. The expressions of TGF-β1, Smad3 and Smad7 proteins in the liver tissue were detected with immunohistochemical staining method. The expressions of TGF-β1, Smad3, Smad7 mRNAs in the liver tussue were detected by RT-PCR. Results The expressions of TGF-β1 (2.59 ± 0.99 vs. 0.43 ± 0.21) and Smad3 (2.56 ± 0.67 vs. 0.41 ± 0.18) proteins and TGF-β1 mRNA (2.25 ± 0.21 vs. 0.71 ± 0.09) and Smad3 (2.34 ± 0.03 vs. 0.78 ± 0.12) mRNAs in the model group were significantly increased than those in the normal control group (all P<0.01). Compared with the model group, the expressions of TGF-β1 (1.12 ± 0.27 vs. 2.59 ± 0.99) and Smad3 (1.05 ± 0.34 vs. 2.56 ± 0.67) proteins in the high-dose Ruangan granule group decreased significantly, the expression of Smad7 increased significantly (2.33 ± 0.62 vs. 0.36 ± 0.18), and the expressions of TGF-β1 (1.09 ± 0.11 vs. 2.25 ± 0.21) and Smad3 (1.10 ± 0.02 vs. 2.34 ± 0.03) mRNAs decreased significantly, the expression of smad7 mRNA (1.18 ± 0.13 vs. 0.38 ± 0.11) increased significantly (P<0.05). Conclusions Ruangan granule can regulate the TGF-β1/Smads signaling pathway via down-regulation of TGF-β1, Smad3 and up-regulation of Smad7 in liver fibrosis in rats.
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@#Objective To explore the establishment of a rat model of acute radiation-induced liver injury and sig-nificance of the dynamic changes of TGF-β1 expression.Methods Forty healthy 6-week old male SD rats were randomly divided into model group (n=30) and control group (n=10).The right liver of rats in the model group was given a single dose of 25 Gy 6 MV X-ray irradiation.Histopathological examination using HE staining and transmission electron microsco-py were conducted to observe the liver pathological changes in rats at 3, 5, and 10 days after irradiation, serum TGF-β1 was detected, and relevant indicators of liver function ( ALT, AST, ALP) were determined.Statistical analysis was per-formed using SPSS 17.0 software.Results At 3, 5 and 10 days after irradiation, early pathological changes in the liver cells were observed by electron microscopy, the expression of TGF-β1 was gradually increased with the time prolongation, and significant differences were found between the model group and the control group at different time points (P<0.05). The light microscopic observation of liver tissues did not show significant differences between the control group and model group.The liver ALT, AST, ALP at different time points did not show significant differences between the two groups ( P>0.05).Conclusion Electron microscopy can be used to evaluate the early changes of radiation-induced liver injury, pri-or to the alterations visible by routine light microscopy.TGF-β1 can be used to predict the degree of radiation-induced liver injury, and may be used as a sensitive serum cytokine in predicting the degree of radiation-induced acute liver injury.
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Objective To investigate the influence of kaempferol on transforming growth factor(TGF)-β1/Smads signal trans-duction in liver tissue of mice with schistosomiasis liver fibrosis. Methods Forty BALB/c mice were randomly divided into a normal control group(8 mice),a praziquantel group(8 mice ),and 4 praziquantel+kaempferol groups with different kaempfer-ol dosages(5,10,15,20 mg/kg respectively,6 mice each group). Besides the normal control group,all the mice in the other 5 groups were infected with Schistosoma japonicum. After the infection for 6 weeks,the praziquantel group and the 4 praziquantel+ kaempferol groups were treated with praziquantel 500 mg/(kg · d) for 2 d,then the mice in the praziquantel group were drenched with normal saline for 6 weeks,and those in the 4 praziquantel+kaempferol groups were drenched with kaempferol 5, 10,15,20 mg/kg respectively for 6 weeks. After the treatment,all the animals were sacrificed by the cervical dislocation meth-od,and the area of egg granuloma and the degree of fibrosis in the livers of the mice were observed by HE and Masson staining. The expressions of TGF-β1,Smad2/3,Smad7 proteins were measured by the immunohistochemical method,and the mRNA lev-els of the 3 proteins were detected by RT-PCR. Results Compared with the mice in the praziquantel group,the areas of egg granuloma of the liver of the mice in the 4 praziquantel+kaempferol groups were smaller,and the degrees of the hepatic fibrosis of the mice were lesser,and their expressions of Smad2 and Smad3 at protein and their mRNA levels were significantly lower (all P<0.05),while the expression of Smad7 at protein and its mRNA level were significantly higher(all P<0.05). Conclu-sion By decreasing the expressions of TGF-β1 and Smad2/3,and increasing the expression of Smad7,kaempferol can signifi-cantly reduce the degrees of hepatic fibrosis and granuloma caused by schistosome eggs after the praziquantel treatment.
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Objective To investigate the effects ofShipi-Gushen-Huayu Recipe on the expressions of collagen I, laminin(LN), transforming growth factor-β1(TGF-β1)andα-smooth muscle actin(α-SMA)in adriamycin-induced renal fibrosis in rats.Methods A total of male SD rats were randomly divided into four groups, with 10 rats in each group: a normal group, a model group, a treatment group and a fosinopril sodium group. Except the rats in the normal group, the rest rats were subjected to renal fibrosisvia tail intravenous injection of adriamycin(4 mg/kg). Two weeks after modeling, the rats in the rreatment group and in the fosinopril sodium group were intragastrically administrated daily withShipi-Gushen-Huayu Recipe extract(43 g/kg)and fosinopril solution(2 mg/kg), respectively,both in the normal group and model group with saline. After 30 days, 24-hours urine protein were determined, and the expressions of collagen I, LN, TGF-β1 andα-SMA in kidney tissue were detected with immunohistochemistry staining.Results The expressions of collagen I(24.64±0.67vs. 32.86±0.88), LN(18.71±0.72vs. 28.35±0.87), TGF-β1(14.71±0.68vs. 18.35±0.96)andα-SMA(17.64±0.74vs. 25.86±0.85)in the treatment group were significantly lower than those in the model group(allP<0.01). The expressions of collagen I, LN, TGF-β1 andα-SMA in the fosinopril sodium group were 27.33±0.73, 20.44±0.81, 15.44±0.85 and 19.33±0.77, respectively. There was no statistically significant difference between the expressions of collagen I, LN, TGF-β1 andα-SMA in the treatment group and in the fosinopril sodium group.ConclusionShipi-Gushen-Huayu Recipe can significantly down regulate the expressions of collagen I, LN, TGF-β1 andα-SMA in adriamycin-induced renal fibrosis in rats.
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[ABSTRACT]AIM:ToevaluatetheeffectsofantisenseTGF-β1oligodeoxynucleotide(ASTGF-β1)ontheex-pression of TGF-β1 , deposition of extracellular matrix ( ECM) and the neointima formation in the arteries after balloon inju-ry.METHODS:The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed.At the same time, sense TGF-β1 oligodeoxynucleotide ( S TGF-β1 ) with the base sequence complement to AS TGF-β1 was synthesized as a control . The oligodeoxynucleotides were introduced into in vivo and in vitro experiments , respectively .RESULTS:The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner , and S TGF-β1 did not have the same effect.Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was ob-served.Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis.Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs . Fibronectin ( FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (0.01~1 μmol/L)-dependent manner .AS TGF-β1 significantly increased the mRNA expression of contractile marker SM 22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla , especially at the concentration of 0.01μmol/L and 0.1 μmol/L.After treatment with AS TGF-β1 (90 μg· kg-1 · d-1 ) for 28 d, the neointima formation was significantly inhibited , and the area ratio of intima/media was markedly decreased by 68% compared with untreated group , but S TGF-β1 had no effect on neointimal formation .CONCLUSION:The AS TGF-β1 specifically inhibits the pro-tein expression of TGF-β1 in the VSMCs derived from injured arteries .Moreover , it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN .Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury .The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the altera-tion of VSMC phenotype after balloon injury .
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Objective To investigate the effect of extract ofTangshen-HuazhuoRecipe(TSHZR) on the serum concentrations of transforming growth factorβ1(TGF-β1) and platelet derived growth factor(PDGF) in patients withⅣ stage of diabetic nephropathy(DN).Methods From June 2012 to December 2012, 98 patients ofⅣstage DN in our hospital outpatient were enrolled and randomly divided into treatment group(n=48) and control group(n=50) using random number table. All patients received conventional therapies of controlling blood sugar, lipid, blood pressure and anticoagulant therapy. On such basis, the control group was treated by irbesartan, 150 mg/d, and the treated group treated by TSHZR combined with irbesartan,150 mg/d, for 6 months. Serum TGF-β1 and PDGF were determined with ELISA before and after treatment,and urinary albumin excretion rate,HbA1c,serum creatinine,blood urea nitrogen and lipid profiles were examined as well. ResultsIn the treated group, the TGF-β1 was(172.5±31.3), (123.6±21.2)pg/ml, the PDGF was(860.9± 131.2), (500.6±130.2)pg/ml before the treatment and after the treatment, respectively. The TGF-β1 and PDGF after the treatment were significantly decreased than those before the treatment(P<0.01). After the treatment, TGF-β1 and PDGF in the treated group were statistically significant compared to the control group[TGF-β1 is(157.4±39.6)pg/ml, PDGF is(765.7±161.8)]pg/ml,P<0.01). After the treatment, the treatment group was superior to the control group in TG(1.72±0.25)mmol/L,(2.09±0.27)mmol/L,(P<0.01), TC(4.56± 0.64)mmol/L,(6.11±0.93)mmol/L, (P<0.01), HDL-C(1.56±0.50)mmol/L,(1.36±0.44)mmol/L, (P<0.01), LDL-C(2.46±1.08)mmol/L(3.32±0.87)mmol/L,(P<0.05)and UAER(100.73±204.24)μg/min, (226.24±396.38)μg/min, (P<0.01).Conclusion TSHZR can inhibit the progressive of IV stage of diabetic nephropathy by suppressing TGF-β1 and PDGF expression level.
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Objective To investigate the effects of transforming growth factorβ1(TGFβ1)and β3 (TGFβ3)gene transfer on MMP-2,MMP-9 and TIMP-1 expression in hepatic stellate cells(HSC-T6).Methods TGFβ1 and TGFβ3 expression plagmids were constructed.The recombinant expression plasmid pcDNA3.1(+)-=TGFβ1 and pcDNA3.1(+).TGFβ3 were transfected or cotransfected into HSC-T6.At 24,48 and 72 h after transfection,the expression of MMP-2,MMP-9 and TIMP-1 mRNA were detected by real-time quantitative PCR,and the expression of MMP-2,MMP-9 and TIMP-1 protein were detected by Western blot.The recombinant expression plasmid pcDNA3.1(+).TGFβ1 was transfected into HSC-T6,and positive clones were selected by G418.The positive clones were transfected by the recombinant expression plasmid pcDNA3.1(+).TGFβ1,and the expression of MMP-2,MMP-9 and TIMP-1 were detected at 48 h after transfection.Results After transfection with peDNA3.1-TGFβ1,MMP-2 and TIMP-1 increaged remarkably in HSC-T6 cells(P＜0.05),but MMP-9 remained at the sanle level;After transfection with pcDNA3.1-TGFβ3,expression levels of MMP-2,MMP-9 and TIMP-1 mRNA were not changed,but TIMP-1 protein increased remarkably(P＜0.05);in cotransfection group,the expression of MMP-2 was higher than that in the blank and the control groups(P＜0.05),but MMP-9 level was not changed and TIMP-1was decreased compared with that in the TGF-β1 transfection group(P＜0.05).After TGFβ3was transfected into positive clones,the change of MMP-2 wag not significant(P＞0.05).but MMP-9 increaged and TIMP-1 decreased significantly at 48 h after transfection(P＜0.05).Conclusions TGFB3 may inhibit liver fibrosis by increase the activity of MMP-2 and MMP-9,and decrease the activity of TIMP-1.
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The expression of Smad2 and Smad3 and the influence of exogenous transforming growth factorβ1 (TGFβ1) on them in rat bone marrow-derived mesenchymal stem cells (MSCs) cultured in vitro were investigated. The effects of different concentrations of TGFβ1 on cell proliferation and ALP activity were detected by MTT and PNPP in MSCs respectively. The expression of Smad2 and Smad3 and the influence of exogenous TGFβ1 on them were also examined by immunocytochemistry and Western blot assays. The exogenous TGFβ1 induced a dose-dependent decrease in cell proliferation and a dose-dependent increase in ALP activity, which plateaued at 5 ng/ml. Smad2 and Smad3 proteins were detected only in the cytoplasm in the absence of TGFβ1 and TGFβ1 could stimulate the translocation of them from the cytoplasm to the nucleus. The total amount of Smad2 protein remained unchanged before and after TGFβ1 treatment (P＞0.05). The expression levels of Smad3 remained unchanged after 3 h and 6 h treatment (P＞0. 05), but decreased markedly after 24 h treatment (P＜0.05). It was concluded that TGFβ1 is a latent osteoinductive factor involved in osteoblastic differentiation. Both Samd2 and Smad3 mediate TGFβ1 signaling as downstream mediators in MSCs. The biological output of TGFβ1 triggering the osteoblastic differentiation could be entirely determined by Smad3 in MSCs.