Expression of connective tissue growth factor in the renal tissue of rats with unilateral ureteral obstruction

AIM: To detect the expression of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in rat unilateral ureteral obstructive (UUO) nephropathy animal model, and to observe the kinetic changes at different stages of firosis. METHODS: Male SD rats were subjected to either left ureteral ligation or sham operation, then killed at 3, 7, 14, 21 or 28 days after UUO or sham operation (n=6 at each time point). HE, Masson or PAS staining were applied to the renal tissue sections. The extent of tubulointerstitial injury was determined by Banff classification. RESULTS: The extent of tubulinterstitial fibrosis became serious with the time of obstruction. Tubules were mostly atropic and replaced by proliferative fibrous tissue at day 28. The expression of CTGF and α-SMA were consistent with the damage of tubulointerstitial. The positive correlation among CTGF or α-SMA and the tubulointerstitial injury scores were significant. The expression of TGF-β1 came to peak at day 7 to 14, and gradually decreased at day 21 and 28. CONCLUSION: These results indicate that the expression of CTGF may be upregulated by TGF-β in UUO rats, and CTGF may be involved in tubulointerstitial fibrosis through the development of myofibroblasts.     

Role of miR-219 and TGFBR2 in pathogenesis of renal fibrosis

AIM:To study the role of microRNA-219 (miR-219) in regulation of transforming growth factor-β receptor type 2 (TGFBR2) in renal fibrosis. METHODS:The renal fibrosis patients (n=70) were selected in this stu-dy, and 20 cases of healthy people were selected as control group. RT-qPCR was used to detect the expression of miR-219 in the serum of the patients with renal fibrosis and control group, and the expression of miR-219 in NRK49F cells after stimulation with angiotensin Ⅱ(AngⅡ) was detected. The protein expression of α-smooth muscle actin (α-SMA) in the NRK49F cells transfected with miR-219 mimics after stimulation with AngⅡ was determined by Western blot. The potential target gene TGFBR2 of miR-219 was screened and verified by the method of luciferase reporter gene. RT-qPCR and Western blot were used to detected the effect of miR-219 mimics on the expression of TGFBR2 at mRNA and protein levels, and the mRNA expression of α-SMA, connective tissue growth factor (CTGF), type I collagen α1 (COL1A1) and COL3A1 in the NRK49F cells was also detected, respectively. The unilateral ureteral occlusion (UUO) mouse model was established and the expression of miR-219 in the renal tissue was monitored. The morphological change of renal fibrosis was observed in the UUO mice after injection of miR-219, and the mRNA expression levels of COL1A1 and COL3A1 were detected. RESULTS:The expression level of miR-219 in the patients with renal fibrosis was significantly lower than that in control group, and the expression of miR-219 in the UUO mice was decreased significantly (P<0.01). The expression level of miR-219 was significantly decreased in the NRK49F cells after AngⅡ stimulation, and miR-219 mimics inhibited the protein expression of α-SMA(P<0.01). miR-219 mimics had a targeted regulatory effect on TGFBR2 gene, which inhibited the mRNA and protein expression of TGFBR2. miR-219 mimics inhibited the mRNA expression of α-SMA, CTGF, COL1A1 and COL3A1. miR-219 also down-regulated the mRNA expression of COL1A1 and COL3A1 in the UUO mice and inhibited the process of renal fibrosis. CONCLUSION:miR-219 inhibits the development of renal fibrosis by inhibiting the expression of TGFBR2, which may become a new target for the diagnosis and treatment of renal fibrosis.     

Preventive effects of anti-IGFBPrP1 antibody on hepatic fibrosis in mice

AIM: To investigate the preventive effect and mechanism of anti-insulin-like growth factor binding protein related protein 1(IGFBPrP1) antibody on hepatic fibrosis induced by thioacetamide (TAA) in mice.METHODS: Twenty-four male C57BL/6 wild-type mice were randomly divided into 3 groups (n= 8 in each group): normal control group, TAA group (4 weeks) and TAA+anti-IGFBPrP1 antibody group (4 weeks). The morphological changes of liver tissues were observed. The expression levels of α-smooth muscle actin (α-SMA), transforming growth factor beta 1 (TGF-β₁), Smad3, phosphorylated Smad2/3 (p-Smad2/3), fibronectin (FN), collagen I, collagen Ⅲ and IGFBPrP1 were detected by the methods of immunohistochemistry and Western blotting.RESULTS: In TAA group (4 weeks), obvious injury of liver was observed, and the expression levels of α-SMA, TGF-β₁, Smad3, p-Smad2/3, FN, collagen Ⅰ, collagen Ⅲ and IGFBPrP1 were significantly increased as compared with normal control group (P<0.01). Compared with TAA group (4 weeks), the injury of the liver was alleviated and the expression levels of the proteins above were decreased in TAA+anti-IGFBPrP1 antibody group (4 weeks, P<0.01). IGFBPrP1 was positively correlated with TGF-β₁, Smad3, p-Smad2/3, FN and collagen I (P<0.01). CONCLUSION: Anti-IGFBPrP1 antibody prevents TAA-induced hepatic fibrosis in mice by inhibiting the activation of hepatic stellate cells, reducing the expression of p-Smad2/3 and inhibiting the TGF-β₁/ Smad3 signal transduction, thereby depressing the deposition of extracellular matrix in liver tissues.     

The role of TGF-β signal protein Smad2/3 in tubulointerstitial fibrosis associated with unilateral ureteral obstruction in rats

AIM:To investigate the functional role of TGF-β₁ signal protein Smad2/3 in tubulointerstitial fibrosis associated with unilateral ureteral obstruction in rats. METHODS:The unilateral ureteral obstruction (UUO) model was induced by the ligation of left ureter. Rats were sacrificed at 1, 3, 7, 14, 21, and 28 days after UUO was initiated. TGFβ₁ protein, phosphorylated Smad2/3 and interstitial α-smooth muscle actin (α-SMA) expression were assayed by immunohistochemical staining. TGF-β1 mRNA in the obstructed kidney was analyzed with in situ hybridration. HE and Masson staining were used for histological and morphometric studies of the pathological change in obstructed kidney. RESULTS:The results showed that upregulation of TGF-β₁ in tubulointerstitium of both cortex and medulla at day 3 (a 3.1 fold increase vs control, P<0.05) when interstitial volume started to increase significantly. The highest expression of TGF-β1 was detected at day 7 (6.2 folds vs control, P<0.01). Phosphorylated Smad2/3, mainly detected in the nucleus of tubular cells, were also markedly upregulated at day 3 (a 3.5 fold increase vs control, P<0.05), and this was steadily increased by day 7 (7.8 folds vs control, P<0.01). The expression of interstitial α-SMA in both cortex and medulla was evident at day 3 (a 3.8 fold increase vs control, P<0.05) and peaked by day 7 (9.2 folds vs control, P<0.01). The deposit of extracellular matrix (ECM) and interstitial volume in renal cortex and medulla continued to increase until day 28 in obstructed kidney. CONCLUSION:These findings suggest that TGF-β₁ signal protein Smad2/3 may play an important role in tubulointerstitial fibrosis associated with unilateral ureteral obstruction in rats.     

Relationship between rat hepatic fibrosis and regulation of hepatic stellate cell autophagy by microRNA-181a

AIM To observe the changes of liver structure, the levels of transforming growth factor-β1 （TGF-β1）, microRNA-181a, LC3-II/-I, beclin-1 and collagen deposition in hepatic fibrosis （HF） rats induced by carbon tetrachloride （CCl₄）, and the effect of microRNA-181a on autophagy of rat hepatic stellate cells （HSCs） induced by TGF-β1, and to explore the possible mechanism of microRNA-181a in regulating HSC activation and HF. METHODS Wistar rats （n=40） were randomly divided into 5 groups （with 8 in each）： control group （subcutaneous injection of olive oil, 3 mL/kg, twice a week）, and CCl₄-induced HF groups of 2, 4, 6 and 8 weeks （subcutaneous injection of 40% CCl₄, 3 mL/kg, twice a week for 2, 4, 6 and 8 weeks, respectively）. Masson staining was used to evaluate the changes of HF in rats. The levels of TGF-β1 in serum and liver tissue of the rats were measured by ELISA. The level of microRNA-181a in rat liver tissues was detected by RT-qPCR. The protein levels of LC3-II/-I, beclin-1, α-smooth muscle actin （α-SMA）, collagen type I （Col I） and collagen type Ⅲ （Col Ⅲ） in rat liver tissues were measured by Western blot. HSC-T6 cells were transfected with microRNA-181a inhibitor, or pretreated with the autophagy inhibitor 3-methyladenine （3-MA）, before treatment with TGF-β1 to stimulate autophagy. The expression of microRNA-181a, LC3-II/-I, beclin-1, α-SMA, Col I and Col Ⅲ in HSC-T6 cells were determined by RT-qPCR and Western blot. RESULTS The levels of TGF-β1, microRNA-181a, LC3-II/-I ratio and beclin-1 in liver tissues showed an overall trend of increasing with the progression of HF, and microRNA-181a expression showed a positive correlation with autophagy-associated proteins （P<0.01）. MicroRNA-181a level was significantly increased, which was associated with TGF-β1-induced autophagy and activation of HSC-T6 cells.MicroRNA-181a expression was significantly down-regulated in the HSC-T6 cells transfected with microRNA-181a inhibitor, along with suppression of autophagy and cell activation （P<0.01）, which were similar to the effects of 3-MA treatment. CONCLUSION CCl₄ promotes rat HF, the microRNA-181a expression of liver tissue, and autophagy in a time-dependent manner. Reducing the expression of microRNA-181a in HSC-T6 cells inhibits the autophagy of HSCs-T6 cells induced by TGF-β1. The regulation of HSC autophagy by microRNA-181a may be involved in rat HF.     

Expression of TGF-β1 and Smad2/3 in kidney of STZ-induced diabetic nephropathy rats

AIM: To study the role of TGF-β/Smad pathway in the development of renal fibrosis in diabetic nephropathy.METHODS: Rats were induced to diabetic nephropathy by using tail intravenous injection of STZ.The expression of TGF-β₁, Smad2/3 protein and mRNA in kidney were examined at 2, 4, 8 and 16 weeks after STZ induction.CTGF, collagen-Ⅲ, PAI-1 mRNA expression in kidney at 16 weeks of STZ-induced diabetic nephropathy and normal rats were studied by RT-PCR.RESULTS: Weak TGF-β₁, Smad2/3 protein were detected in normal renal tissues while strong TGF-β₁, Smad2/3 staining were observed in renal tissues of diabetic nephropathy (0.057±0.030/0.223±0.040;0.017±0.010/0.153±0.010, respectively, P<0.05).The TGF-β₁, Smad2/3 protein expression were constantly high with the development of diabetic nephropathy and fibrosis (0.153±0.010, 0.122±0.050, 0.141±0.070 and 0.216±0.030 for 2, 4, 8 and 16 weeks, respectively).The TGF-β₁, Smad2 mRNA expression also increased with the development of diabetic nephropathy (2.86, 3.25 fold compared to control, respectively).The expression of TGF-β₁, Smad2, CTGF, collagen-Ⅲ and PAI-1 mRNA were significantly higher in kidney of 16 week diabetic nephropathy rats than that in normal ones (3.92, 2.95, 1.57, 1.95 and 1.97 folds compare to control, respectively, P<0.05).CONCLUSION: The results indicate that TGF-β₁/ Smad2 pathway activity might play an important role in pathophysiological process of diabetic nephropathy.It may be involved in diabetic renal fibrosis through up-regulation of CTGF and PAI-1 to promote extracellular matrix deposition.     

Effect of tumor suppressor phosphatase and tensin homology deleted in chromosome 10 on renal interstitial fibrosis in IgA nephropathy

AIM: To explore the potential correlation between the expression of phosphatase and tensin homology deleted on chromosome 10(PTEN) and RIF in IgA nephropathy. METHODS: Forty-seven patients diagnosed as primary IgA nephropathy by renal histology were involved. 10 specimens from normal renal tissue of renal carcinoma served as control group. Tubulointerstitial lesion (TIL) was classified by using Katafuchi scale, including no TIL (group I), mild TIL (group II), moderate TIL (group III), severe TIL (group IV). The expressions of PTEN, TGF-β1, α-SMA and ColⅢ in renal tissue were detected by immunohistochemistry. PTEN mRNA was detected by in situ hybridization. RESULTS: Renal tissues from renal biopsy showed abundant expressions of PTEN and PTEN mRNA in endochylema of renal tubular epithelial cells, and negligible expression in glomeruli. With the progress of TIF in IgA nephropathy, the expressions of PTEN and PTEN mRNA decreased gradually (P<0.05). In contrast, the expressions of TGF-β1, α-SMA and ColⅢ increased significantly (P<0.05). The expressions of PTEN and PTEN mRNA in renal tissues were positively with eGFR and urine osmotic pressure (P<0.01), negatively correlated with the expressions of TGF-β1, α-SMA, ColⅢ, urinary protein excretion for 24 h, the rate of sclerosis glomeruli and the scores of vascular lesion (P<0.01). CONCLUSION: These results suggest the loss of PTEN expression in RIF of IgA nephropathy. Moreover, TGF-β signaling induces epithelial to mesenchymal transition and extracellular matrix accumulation possibly through a mechanism dependent on the downregulation of PTEN.     

Kinetics of pathologic changes in bleomycin-induced murine pulmonary fibrosis model

AIM: To Investigate the kinetics of pathologic changes in bleomycin-induced pulmonary fibrosis in rats. METHODS: Sixty male SD rats were randomized as a negative control group and pulmonary fibrosis model groups (B3, B7, B14, B28, B56 sub-groups). Except for control group, rats in the other groups were intratracheally administered with bleomycin. Animals in pulmonary fibrosis model groups were sacrificed on day 3, 7, 14, 28 and 56. The sections of the right lung were stained by HE, Masson and sirius red. The left lung was weighed and its hydroxyproline content was assayed. The mRNAs of TGF-β1, MMP-9 and TIMP-1 in the lung homogenate were measured by semi-quantitative RT-PCR. The expressions of TGF-β1, MMP-9 and TIMP-1 in lungs were observed by immunohistochemistry. RESULTS: (1) The content of lung hydroxyproline in pulmonary fibrosis model groups was significantly increased than that in control group (P<0.05). The pulmonary inflammation in pulmonary fibrosis model groups was significantly serious than that in control group, pulmonary fibrosis in B14, B28 and B56 groups was also significantly serious than that in control group. (2) A small quantity of TGF-β1, MMP-9 and TIMP-1mRNA were measured in normal lung, and the expression increased significantly after administration of bleomycin. Different expressions of TGF-β1, MMP-9 and TIMP-1 in different days after bleomycin administration were observed. CONCLUSION: The pathological changes in different days after bleomycin administration are different. TGF-β1, MMP-9 and TIMP-1 may play important roles in the pathogenesis of pulmonary fibrotic process.     

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.     

Up-regulation of miR-133a expression attenuates myocardial fibrosis in spontaneously hypertensive rats

AIM: To investigate the effect of up-regulated expression of microRNA-133a (miR-133a) on myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Wistar-Kyoto (WKY) rats with homologous normal blood pressure served as the normal control group. SHR were divided into SHR group, SHR+ adeno-associated virus (AAV) group and SHR+miR-133a-AAV group randomly. miR-133a carried by miR-133a-AAV was transfected into SHR heart by coronary perfusion. The rat tail artery pressure was monitored. The myocardial collagen deposition was observed by Masson staining. The expression of miR-133a in myocardial tissue was detected by real-time PCR. The protein levels of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) were determined by immunohistochemistry and Western blot. RESULTS: Compared with the WKY rats, the tail artery pressure of the SHR increased significantly. The expression of miR-133a in heart decreased, and the expression levels of TGF-β1 and CTGF increased (P<0.05), and myocardial fibrosis occurred. After up-regulating the expression level of miR-133a in the heart of SHR, the myocardial fibrosis was significantly reduced, and the expression levels of TGF-β1 and CTGF decreased (P<0.05). CONCLUSION: Up-regulation of the miR-133a expression improves myocardial fibrosis induced by hypertension, which may be related to inhibiting the protein expression of TGF-β1 and CTGF in myocardium.     

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

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

miR-140-3p inhibits viability,invasion and migration of non-small-cell lung cancer A549 cells by targeting PD-L1

AIM: To explore the target relationship between microRNA-140-3p (miR-140-3p) and programmed cell death ligand 1 (PD-L1) and their effect on the viability, migration and invasion of non-small-cell lung cancer A549 cells.METHODS: RT-qPCR was used to detect the miR-140-3p expression in HLF-1, A549 and H1299 cells, and then the A549 cells with the most significant difference were selected as the subsequent research object. TargetScan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between miR-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of miR-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells.RESULTS: miR-140-3p was significantly down-regulated in the A549 cells and H1299 cells (P<0.05). Transfection with miR-140-3p mimic decreased the expression of PD-L1 and inhibited the viability, migration and invasion of the A549 cells. Transfection with pcDNA3.0-PD-L1 reversed the inhibitory effect of miR-140-3p on the viability, migration and invasion of the A549 cells.CONCLUSION: miR-140-3p inhibits the viability, migration and invasion of A549 cells by targeting PD-L1.     

Expression of programmed cell death factor 4 in pulmonary fibrosis model

AIM:To detect the expression of programmed cell death protein 4 (PDCD4) in pulmonary fibrosis model and its effect on cell viability and pulmonary fibrosis indicators, and to explore its mechanism. METHODS:The expression level of PDCD4, α-smooth muscle actin (α-SMA) and collagen type I (COL-I) in human embryonic lung fibroblasts cell line HFL-1 group and HFL-1+TGF-β1 group were detected by Western blot and RT-qPCR. The plasmid pEZ-M03-PDCD4 and empty vector pEZ-M03 were transfected into myofibroblasts (MB), and the protein expression level of PDCD4 was detected by Western blot. The protein levels of p-AKT and AKT in blank control group, pEZ-M03-PDCD4 group, pEZ-M03 group and LY294002 group and the expression of cell cycle-related proteins c-Myc and cyclin D1 were determined by Western blot. The effect of PDCD4 on the viability of MB was measured by CCK-8 assay. The hydroxyproline content in the culture supernatant of HFL-1 cells and transfected MB was detected by hydroxyproline digestion method. The expression of PDCD4 in the lung tissues of the mice in model group and control group was detected by Western blot. RESULTS:Compared with HFL-1 group, the expression of α-SMA and COL-I at mRNA and protein levels in HFL-1+TGF-β1 group was significantly increased (P<0.01), the mRNA expression of PDCD4 was not significantly changed, while PDCD4 protein was significantly down-regulated (P<0.01). Compared with blank control group and pEZ-M03 group, the protein expression of PDCD4 in pEZ-M03-PDCD4 group was significantly increased (P<0.05), the protein expression of c-Myc and cyclin D1 was significantly decreased (P<0.05), the cell viability was also significantly inhibited (P<0.01), and the content of hydroxyproline in the culture supernatant was significantly reduced (P<0.05). Compared with blank control group, the protein levels of p-AKT were significantly decreased in pEZ-M03-PDCD4 group and LY294002 group, and no significant difference between blank control group and pEZ-M03 control group was observed. Compared with control group, PDCD4 expression was decreased in model group (P<0.01).CONCLUSION:PDCD4 is low expressed in the process of pulmonary fibrosis. Over-expression of PDCD4 inhibits the viability of MB, decreases the content of hydroxyproline, and inhibits the PI3K/AKT signaling pathway.     

Up-regulation of miR-181a attenuates releases of CSE-induced pro-inflammatory factors and expression of collagen IV,fibronectin and α-SMA in human bronchial epithelial cells

AIM: To investigate the effect and potential mechanism of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced the productions of pro-inflammatory factors and the expression of collagen IV, fibronectin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (HBECs). METHODS: CSE-induced miR-181a expression was detected by RT-qPCR in the HBECs. After tansfected with miR-181a mimic, the releases of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and transforming growth factor-β1 (TGF-β1) were measured by ELISA, the protein expression of collagen IV, fibronectin and α-SMA was determined by Western blot. The activation of NF-κB/TGF-β1/Smad3 pathway was also evaluated by Western blot. RESULTS: CSE increased the levels of TNF-α, IL-1β, IL-6 and TGF-β1 and the expression of collagen IV, fibronectin and α-SMA, and decreased the expression of miR-181a in the HBECs (P<0.05). However, transfected with miR-181a mimic partially prevented the releases of TNF-α, IL-1β, IL-6 and TGF-β1, and inhibited the expression of collagen IV, fibronectin and α-SMA (P<0.05). Additionally, the activation of NF-κB/TGF-β1/Smad3 evoked by CSE was attenuated after transfected with miR-181a mimic. CONCLUSION: Up-regulation of miR-181a prevents the releases of CSE-induced pro-inflammatory factors and expression of collagen IV, fibronectin and α-SMA in the HBECs, and its mechanism may be related to the inhibition of NF-κB/TGF-β1/Smad3 pathway.     

Role of TGFβ1/Smad3 signaling pathway-mediated lysine hydroxylase 2 activity in collagen deposition of pulmonary fibrosis

AIM: To investigate the effect of TGFβ1/Smad3 signaling pathway on the changes of lysyl hydro-xylase2 (LH2) activity, and to study the role in the relationship between LH2 and collagen deposition of pulmonary fibrosis. METHODS: Human lung fibroblast cell line HFL1 was cultured in F12 medium with 10% fetal bovine serum. The cells were divided into control group, TGFβ1 (10 μg/L) stimulation group, and minoxidil (5 μmol/L) intervention group. The cells in control group were treated with the equivalent volume of medium. The RNA and protein were collected after 48 h. The mRNA levels of PLOD2, α-SMA and COLⅠ were detected by RT-qPCR. The protein levels of LH2, total Smad3, phosphorylated Smad3, α-SMA, COLⅠ and COL Ⅳ were determined by Western blot. Hydroxylysylpyridinoline (HP) content was detected by ELISA. RESULTS: After stimulation with TGFβ1, the mRNA expression of PLOD2, α-SMA and COLⅠ was increased (P<0.01), and the protein levels of LH2, p-Smad3, α-SMA, COLⅠ and COL Ⅳ were also up-regulated, but the total Smad3 protein did not change. Treatment with minoxidil decreased the levels of above indexes (P<0.01). Compared with control group, stimulation with TGFβ1 increased the content of HP. However, treatment with minoxidil decreased the synthesis of HP (P<0.05). CONCLUTION: Activation of TGFβ1/Samd3 signaling pathway enhances LH2 expression. Minoxidil inhibits the TGFβ1/Samd3 signaling transduction, thereby reducing the expression of LH2 and the synthesis of hydroxylysyl collagen pyridine chain, and reducing pulmonary fibrosis.