Role of miRNA-10a in renal ischemia-reperfusion injury in mice: the relationship with TGF-β 1/Smad2 signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of microRNA-10a (miRNA-10a) in renal ischemia-reperfusion (I/R) injury in mice and the relationship with transforming growth factor beta1 (TGF-β 1)/Smad2 signaling pathway. Methods:Twenty-four SPF healthy male adult C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 4 groups ( n=6 each) by the random number table method: sham operation group (S group), renal I/R group (IR group), renal I/R plus miRNA-10a antagonist group (I group), and renal I/R+ miRNA-10a agonist group (M group). The mouse model of renal I/R was developed by clamping the left renal pedicle for 45 min followed by reperfusion in anesthetized animals.miRNA-10a antagonist and agonist 20 nmol were injected via the tail vein once every 24 h for 3 consecutive days starting from 72 h before surgery in group M and group I, respectively, while the equal volume of normal saline was given instead in S and IR groups.Blood samples were collected from the orbital vein at 24 h of reperfusion to determine the concentrations of the serum creatinine (Scr) and blood urea nitrogen (BUN). Then the mice were sacrificed, and the kidney tissues were taken for determination of the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, contents of interleukin-1 beta (IL-β) and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay), and expression of TGF-β 1 and phosphorylated Smad2 (p-Smad2) (by Western blot) and for microscopic examination of the pathological changes.The damage to the renal tubules was scored. Results:Compared with group S, the concentrations of Scr and BUN, contents of MDA, IL-1β and TNF-α in renal tissues and renal tubular damage score were significantly increased in IR, I and M groups, and the activity of SOD was significantly decreased, and the expression of TGF-β 1 and p-Smad2 was up-regulated in IR and M groups ( P<0.05). Compared with group IR, the concentrations of Scr and BUN, contents of MDA, IL-1β and TNF-α in renal tissues and renal tubular damage score were significantly decreased, the activity of SOD was increased, and the expression of TGF-β 1 and p-Smad2 was down-regulated in group I, and the concentrations of Scr and BUN, contents of MDA, IL-1β and TNF-α in renal tissues and renal tubular damage score were significantly increased, the activity of SOD was decreased, and the expression of TGF-β 1 and p-Smad2 was up-regulated in group M ( P<0.05). Compared with group I, the concentrations of Scr and BUN, contents of MDA, IL-1β and TNF-α in renal tissues and renal tubular damage score were significantly increased, the activity of SOD was decreased, and the expression of TGF-β 1 and p-Smad2 was up-regulated in group M ( P<0.05). Conclusions:miRNA-10a is involved in the process of renal I/R injury and is related to activation of TGF-β 1/Smad2 signaling pathway in mice.

The effects of TGF-β1 and Smad2 on liver fibrosis of biliary atresia / 天津医药

Objective To investigate the expression and function of transforming growth factor (TGF)-β1 and Smad2 in liver fibrosis of biliary atresia (BA). Methods Liver biopsy specimens were collected from autopsy (normal group, n=5), congenital biliary dilatation (CBD group, n=10), BA patients underwent Kasai procedure (early hepatic fibrosis group, n=19) and liver transplantation (transplantation group, n=11). The first three groups were collected from January 2010 to July 2014 in Tianjin Children’s Hospital, and the last group was collected from January 2013 to January 2014 in Tianjin First Central Hospital. The hematoxylin and eosin (HE) stain were used to observe the degree of liver fibrosis of four groups. Immunohistochemistry (IHC) was used to observe expressions of TGF-β1 and Smad2 in liver tissues of these samples. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the quantitative mRNA of TGF-β1 and Smad2 in these samples. Results Results of HE showed that no fibrosis in autopsy group, mild fiber cell hyperplasia in CBD group, severe fibrosis in Kasai group and significant pseudolobule in transplantation group. Results of IHC showed that TGF-β1 was expressed in the cytoplasm of hepatocytes, bile duct cells, lymphocytes and neutrophils. The average optical density of TGF-β1 was the highest in Kasai group compared with that of other three groups (P 0.05). Results of qRT-PCR showed that both TGF-β1 mRNA and Smad2 mRNA were the highest in early hepatic fibrosis group than those of CBD group and transplantation group (P<0.017). Conclusion In early stage of BA, TGF-β1 and Smad2 promote liver fibrosis until the formation of P-P,P-C desmosome structure. However, with BA fibrosis becomes more serious, the pro-fibrogenic function of TGF-β1 and Smad2 becomes less.

Inhibitory effect of colchicine on transforming growth factor β1/Smads pathway in rat models of chronic pancreatitis / 中国组织工程研究

BACKGROUND:Pancreatic stelate cels transforming growth factor β1/Smads signaling pathway activation is probably a main molecular mechanism of pancreatic fibrosis. If this pathway can be blocked, the progression of fibrosis of tissues with chronic pancreatitis wil be inhibited. OBJECTIVE:To study the inhibitory effect of colchicine on transforming growth factor β1/Smads pathway in chronic pancreatitis rat models. METHODS:Healthy male Sprague-Dawley rats were randomly divided into colchicines-treated group and chronic pancreatitis group. After successful establishment of rat models of chronic pancreatitis, the rats in the colchicines-treated group were intraperitonealy injected with colchicine 150 μg/kg daily. The rats in the chronic pancreatitis group were intraperitonealy injected with equal volume of physiological saline daily. Pancreatic tissues were colected after 3 months. Hematoxylin-eosin staining was used to observe histopathological changes of pancreatic tissue. Immunohistochemical staining was used to detect the expressions of transforming growth factor β1 in pancreatic tissue. Western blot assay was utilized to detect the expressions of P-Smad2, P-Smad3 and α-SMA protein in pancreatic stelate cels. RESULTS AND CONCLUSION: Hematoxylin-eosin staining results revealed that compared with the colchicines-treated group, glandular tissue had reduced, while fibrous connective tissue and inflammatory cels had increased obviously and replaced the pancreatic gland tissue in the chronic pancreatitis group. Immunohistochemical staining results demonstrated that the expression levels of transforming growth factor β1 and the index of positive cels were significantly lower in the colchicines-treated group than those in the chronic pancreatitis group (P < 0.05). Western blot assay results revealed that the results of P-Smad2/β-actin, P-Smad3/β-actin andα-SMA/β-actin in pancreatic stelate cels were significantly lower in the chronic pancreatitis group than those in the colchicines-treated group (P < 0.05). Results suggested that colchicine could inhibit the activity of transforming growth factor β1/Smads pathway and pancreatic tissue fibrosis in chronic pancreatitis rats. Therefore, colchicine can be used as a new candidate therapeutic scheme for chronic pancreatitis fibrosis.

miR-18a-5p MicroRNA Increases Vascular Smooth Muscle Cell Differentiation by Downregulating Syndecan4

BACKGROUND AND OBJECTIVES: Differentiation and de-differentiation of vascular smooth muscle cells (VSMCs) are important events in atherosclerosis and restenosis after angioplasty. MicroRNAs are considered a key regulator in cellular processes such as differentiation, proliferation, and apoptosis. Here, we report the role of new miR-18a-5p microRNA and its downstream target genes in VSMCs and in a carotid balloon injury model. MATERIALS AND METHODS: Expression of miR-18a-5p and its candidate genes was examined in VSMCs and in a carotid artery injury model by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and microRNA microarray analysis. VSMC differentiation marker genes including smooth muscle (SM) alpha-actin and SM22alpha were determined by Western blot, qRT-PCR, and a SM22alpha promoter study. Gene overexpression or knockdown was performed in VSMCs. RESULTS: miR-18a-5p was upregulated in the rat carotid artery at the early time after balloon injury. Transfection of the miR-18a-5p mimic promoted the VSMC differentiation markers SM alpha-actin and SM22alpha. In addition, miR-18a-5p expression was induced in differentiated VSMCs, whereas it decreased in de-differentiated VSMCs. We identified syndecan4 as a downstream target of miR-18-5p in VSMCs. Overexpression of syndecan4 decreased Smad2 expression, whereas knockdown of syndecan4 increased Smad2 expression in VSMCs. Finally, we showed that Smad2 induced the expression of VSMC differentiation marker genes in VSMCs. CONCLUSION: These results indicate that miR-18a-5p is involved in VSMC differentiation by targeting syndecan4.

The Role of Expression of TGF-βand Smad Signaling Pathway andα-SMA in Patients with Diabetic Nephropathy / 天津医药

Objective To investigate the mechanism of the pathogenesis of diabetic nephropathy (DN) by detecting the expressions of transforming growth factor (TGF)-β, Smad proteins andα-smooth muscle actin (α-SMA) in kidney biopsy of patients with diabetic nephropathy (DN). Methods Twenty-eight patients with DN who underwent renal biopsy were col-lected as DN group. Ten subjects without DN who underwent nephrectomy were taken as control group. The expressions of TGF-β1, TGF-βRⅠ,TGF-βRⅡ,Smad2/3 andα-SMA in renal tissues were detected by immunohistochemistry stain. Results (1)TGF-β1,TGF-β RⅠ,TGF-β RⅡ,and Smad2/3 were expressed in the glomeruli and tubules of both DN group and control group, while the expressions of TGF-βand Smad proteins were significantly higher in DN group than those in control group. At the early stage of DN, TGF-βand Smad proteins were significantly expressed even though there was no remarkable lesions observed by light microscopy. There was no correlation between increased expression and the progres-sion of DN. These proteins were not expressed after glomerulus and renal tubule fibrosis. (2) In control group,α-SMA was identified only in the vascular walls, glomerulus and renal tubules, while it was expressed in almost all parts of kidney in DN group. Conclusion TGF-β and Smad signals involved in the pathogenesis of DN, which may have similar pathogenesis with immune complex glomerulonephritis.

Regulation of p53 expression though transforming growth factor β1 (TGFβ1)/Smads pathway in actinic keratosis / 中华皮肤科杂志

Objective To evaluate the performance of actinic keratosis (AK) tissue as a culture model for the study of interference in transduction pathway,and to explore the mechanism underlying the p53 regulation though TGFβ1/Smads pathway by using the tissue culture model.Methods Twenty-five skin samples from the lesions of patients with AK were cultured,and divided into 5 groups to be treated with TGFβ1 of 10 μg/L for 24 and 48 hours,the tran sforming growth factor (TGF) β1 receptor kinase inhibitor SB431542 of 10 μmol/L for 24 and 48 hours,respectively,or remain untreated.Real time PCR and Western blot were performed to quantify the mRNA expression of p53 and protein expression of p53 and phosphorylated Smad2 in these tissue specimens respectively.Results A significant elevation was observed in the expressions of p53 mRNA ( 13.4968 ± 0.9903 vs.1,P ＜ 0.05) and phosphorylated Smad2 (0.700 ± 0.023 vs.1,P ＜ 0.05) in AK tissues after treatment with TGFβ1 for 24 hours,and in the expressions of p53 mRNA (13.3882 ± 1.6772 vs.1,P ＜ 0.05) and protein (1.009 ± 0.001 vs.0.512 ± 0.005,P ＜ 0.05) after treatment with TGFβ1 for 48 hours,compared with the untreated AK tissues.No significant differences were observed in the expression of p53 protein between the AK tissues treated with TGFβ1 for 24 hours and 48 hours (P ＞ 0.05).SB431542 induced a statistical reduction in the level of phosphorylated Smad2 at 48 hours (0.116 ± 0.003 vs.0.306 ± 0.023,P ＜ 0.05),but no significant changes were observed in the expression of p53 mRNA or protein after SB431542 treatment for 24 or 48 hours.Conclusions AK tissue cultures can serve as a model for the study of interference in signal transduction pathway.TGFβ1 might regulate the expression of p53 protien through Smads pathway in AK.

Effect of propofol on transforming growth factor-β1/Smad2 signaling pathway in lung tissues in rats with lipopolysaccharide-induced acute lung injury / 中华麻醉学杂志

Objective To investigate the effect of propofol on transforming growth factor (TGF)-β1/Smad2 signaling pathway in lung tissues in rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI).Methods Fifty-six male Wistar rats,aged 7-8 weeks,weighing 260-300 g,were randomly divided into 5 groups:control group (group A,n =8) ; LPS group (group B,n =12); 3 propofol groups (groups C,D,E,n =12).ALI was induced by intravenous LPS 8 mg/kg in groups B,C,D and E.In groups C,D,E,propofol 5 mg/kg was injectedintravenously before LPS administration and at 0 and 1 h after LPS administration,respectively,followed by infusion of propofol at 10 mg· kg-1 · h-1 until 5 h after LPS administration.Group A received the equal volume of normal saline.Arterial blood samples were collected immediately before LPS administration and 1,3 and 5 h after LPS administration for determination of pH value and PaO2.Then the animals were sacrificed and the lungs were immediately removed for calculation of the wet/dry lung weight ratio and for determination of the expression of TGF-β1-mRNA and Smad2 in lung tissues.Results Compared with group A,pH value and PaO2 were significantly decreased,wet/dry lung weight ratio was increased and the expression of TGF-β1 mRNA and Smad2 was up-regulated in groups B and E (P ＜ 0.05).Compared with group B,pH value and PaO2 were significantly increased,wet/dry lung weight ratio was decreased and the expression of TGF-β1 mRNA and Smad2 was down-regulated in groups Cand D,and PaO2 was significantly increased in group E (P ＜ 0.05).Conclusion The mechanism by which propofol alleviates ALI induced by LPS is related to inhibition of TGF-β1/Smad2 signaling pathway.

The expression and significance of Smad2 in cervical cancer tissue / 中国基层医药

ObjectiveTo explore the expression of Smad2 in cervical carcinoma and analyze the clinical significance of Smad2.MethodsThe expression of Smad2 in normal uterus group( NE group,n =61 ) and cervical carcinoma group( CC group,n =53 ) were detected by immunohistochemistry to analyze the significance of Smad2 in the cervical carcinoma.ResultsPositive rate of expression of Smad2 protein in NE group was 39.3％,positive rate was 77.4％ in CC group,statistical difference was found between the two groups(P ＜ 0.01 ).The degree of differentiation of cervical cancer was lower,and the Smad2 positive rate was higher,well-differentiated and poorly differentiated were 52.6％ and 91.2％,respectively,and the difference was statistically significante ( P ＜ 0.01 ) ; The invasion of cancer was higher,and the Smad2-positive rate was higher ≤ 1/2 and ＞ 1/2 were 66.7％ and 91.3％,respectively,and the difference was statistically significante ( P ＜ 0.05 ).ConclusionThe expression of Smad2 was less; Smad2 was related to depth of invasion and degree of differentiation of cervical carcinoma,which could be used as clinical diagnosis and prognostic markers.