Protective effect of decorin on acute ischaemia-reperfusion injury in the rat kidney

Introduction

Transforming growth factor-β1 (TGF-β1) has a crucial role in collagen synthesis and fibrosis. TGF-β1 can be antagonized and/or reduced by the action of certain agents. We propose to identify the role of decorin in treatment of tubular and interstitial fibrosis and in the inhibition of TGF-β1 in an acute ischaemic kidney.

Material and methods

We grouped 34 female Sprague Dawley type rats into 3 groups as 9 sham, 9 ischaemia-reperfusion (I/R) and 16 I/R + decorin respectively. The rats in the I/R + decorin group had decorin administered intraperitoneally at the dose of 0.1 mg/kg for 9 days after reperfusion. After 9 days, all the rats in the 3 groups were unilaterally nephrectomized. The TGF-β1 level was measured immunohistochemically in the nephrectomized material.

Results

The TGF-β1 level was lower in the I/R + decorin group. Evaluation of apoptotic activity level by caspase staining showed a statistically significant difference between the 3 groups. The number of caspase stained cells was lower in the I/R + decorin group. The amount of collagen in interstitial tissue was higher in the I/R group than in the I/R + decorin group, but this difference was not statistically significant.

Conclusions

We found that the TGF-β1 level – the so-called initiator of fibrotic activity – and apoptotic activity were low in the I/R + decorin group. Additional studies must be performed to understand the role of decorin in inhibition of TGF-β1 and to assess decorin’s routine use in acute renal ischaemia.     

Effects of rhBNP on myocardial fibrosis after myocardial infarction in rats

Purpose: We aimed to observe the effects and mechanism of rhBNP treatment on myocardial fibrosis (MF) after myocardial infarction (MI). Methods: SPF rats were separated into 3 groups: normal, MI (ligation of left coronary artery), and MI + rhBNP (recombinant human brain natriuretic peptide). Rats in MI + rhBNP group were given 30 μg/kg for 2 days before modeling and for 4 weeks after modeling. mRNA levels and the expression levels of TGF-β1 (transforming growth factor) and CTGF (connective tissue growth factor) in 3 groups were analyzed using the RT-qPCR and western blotting analysis, respectively. Furthermore, myocardial volume fraction (CVF) was analyzed using the Sirius Red F3B (SR) while the percentage of type I and III collagen in 3 groups were analyzed using the immunohistochemical staining. Results: Compared with the normal group, the levels of TGF-β1, CTGF, CVF, type I and III collagen were higher in MI group. However, mRNA levels of TGF-β1 and CTGF were significantly decreased in MI + rhBNP compared to MI groups. Expression of TGF-β1 was lower while that of CTGF was higher in MI + rhBNP group than that in MI group. Besides, CVF, and type I and III collagen were lower in MI + rhBNP group compared with MI group. Conclusion: rhBNP could significantly decrease the TGF-β1 and CTGF levels in post-MI so as to inhibit the type I and III collagen deposition in MF of post-MI. rhBNP will be benefit for the improvement of MF.     

Astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissues of diabetic KKAy mice

Numerous cytokines participate in the occurrence and development of inflammation and renal interstitial fibrosis. Previous studies confirmed that TGF-β1 overexpressed in diabetic nephropathy. As a downstream signal protein of TGF-β1 family, SMAD has an important role in the process of α-SMA mediated renal interstitial fibrosis. This study aimed to study astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissue of diabetic KKAy mice, to reveal its potential impact on renal interstitial fibrosis. 20 type II diabetic KKAy mice were randomly equally divided into model group and astragaloside group, while 10 male C57BL/6J mice were selected as the control. Astragaloside at 40 mg/(kg•d) was given when the KKAy mice fed with high-fat diet to 14 weeks old. The mice were killed at 24 weeks old and the kidney tissue samples were collected. Pathology morphological changes were observed. TGF-β1, SMAD2/3, and α-SMA expression levels were determined by immunohistochemistry. Compared with control, mice kidney in model group appeared obvious fibrosis and up-regulated blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression (P < 0.05). Mice in astragaloside group exhibited alleviated renal interstitial fibrosis compared with the model. Its blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression levels were significantly lower than the model group (P < 0.05). Astragaloside can delay the renal fibrosis process in diabetic mice by influencing the TGF-β/SMADS signaling pathway and down-regulating TGF-β1, SMAD2/3, and α-SMA expression.     

Nox2 contributes to cardiac fibrosis in diabetic cardiomyopathy in a transforming growth factor-β dependent manner

Purpose: This study aimed to investigate the effect of Nox2 on cardiac fibrosis and to elucidate the regulatory mechanism of Nox2 in the development of DCM. Methods: We established normal and insulin-resistant cellular model using neonatal rat cardiac fibroblasts. Then Nox2-specific siRNA were transfected into cardiac fibroblasts with Lipofectamine ® 2000 and crambled siRNA sequence was considered as control. Meanwhile, a part of cells were randomly selected to be treated with or without transforming growth factor-β (TGF-β). Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were respectively performed to determine the expression level of related molecules, such as Nox2, collagen type I and III (COL I and III) and PI3K/AKT and PKC/Rho signaling pathway-related proteins. Results: TGF-β stimulation significantly increased the expression level of Nox2 both in mRNA and protein levels. Suppression of the Nox2 markedly decreased the expression of COL I and COL III in normal and insulin-resistant cellular model with TGF-β stimulation. Moreover, suppression of the Nox2 significantly decreased the expression of PI3K/AKT and PKC/Rho signaling pathway-related proteins in insulin-resistant cellular model with TGF-β stimulation. However, suppression of Nox2 had no effects on these proteins without TGF-β stimulation. Conclusions: Our finding reveals that Nox2 may promote synthesis of COL I and III via involved in PI3K/AKT and PKC/Rho signaling pathway in a TGF-β dependent manner and consequently promote cardiac fibrosis in the development of DCM.     

Correlation between platelet-derived growth factor signaling pathway and inflammation in desoxycorticosterone-induced salt-sensitive hypertensive rats with myocardial fibrosis

Objective: To investigate whether inflammation could excessively activate platelet-derived growth factor (PDGF) signaling pathway in desoxycorticosterone (DOCA) induced salt-sensitive hypertensive rats with myocardial fibrosis (MF). Methods: A total of 30 male SD rats underwent right nephrectomy and then bred with 1% sodium chloride and 0.1% potassium chloride for 2 weeks. These animals were randomly divided into 3 groups: CON group, DOCA group and DOCA+FAS group. Systolic blood pressure (SBP) was measured once every 2 weeks; HE staining was done to observe myocardial inflammation; immunohistochemistry was done to detect expressions of monocyte-macrophage antigen (ectodermal dysplasia 1, ED-1), PDGFRα and PDGFRβ in the myocardium; real time fluorescence quantitative PCR was employed to detect the mRNA expressions of DGF-A, PDGF-B, PDGF-C, PDGF-D, PDGFRα and PDGFRβ. Results: The SBP in DOCA group and DOCA+FAS group increased markedly when compared with CON group (P<0.01), but there was no marked difference between DOCA group and DOCA+FAS group (P>0.05). At 14 days, in DOCA group, the myocardial inflammation was obvious, ED-1 expression increased markedly, the mRNA expressions of PDGF-A, PDGF-B, PDGF-C, PDGFRα and PDGFRβ increased to different extents, protein expressions of PDGFRα and PDGFRβ also elevated markedly (P<0.01), but the PDGF-D mRNA expression remained unchanged, when compared with CON group. After treatment with fasudil (a drug with anti-inflammatory activity), myocardial inflammation was significantly attenuated, mRNA expressions of PDGF-A, PDGF-B, PDGF-C and PDGFRα as well as PDGFRα protein expression reduced dramatically (P<0.01), but the mRNA and protein expressions of PDGFRβ remained unchanged (P>0.05) when compared with DOCA group. Conclusion: In DOCA/salt induced hypertensive rats with MF, excessive activation of PDGF/PDGFR signaling pathway is involved in myocardial inflammation.     

Role of PDGFs/PDGFRs signaling pathway in myocardial fibrosis of DOCA/salt hypertensive rats

This study aimed to investigate the role of PDGF/PDGFR signaling pathway in myocardial fibrosis of desoxycorticosterone (DOCA) induced salt-sensitive hypertensive rats and explore the influence of PDGF/PDGFR signaling pathway on fibroblasts and myofibroblasts in the heart. 60 male SD rats underwent right nephrectomy and bred with 1% sodium chloride and 0.1% potassium chloride for 4 weeks, and then randomly divided into 3 groups (CON group, DOCA group and DOCA+IMA group). Results showed that: 1) 14 and 28 days after intervention, the SBP in DOCA and DOCA+IMA group was significantly higher than that in CON group. At days 28, the severity of myocardial fibrosis and PVCA/VA ratio in DOCA group were significantly increased when compared with CON group. The severity of myocardial fibrosis and PVCA/VA ratio in DOCA+IMA group were markedly lower than those in DOCA group although they were higher than those in CON group. 2) At days 14, the mRNA expressions of PDGFRα and PDGFRβ in DOCA group were significantly higher than CON and DOCA+IMA group. At days 28, the mRNA expressions of PDGFRβ, FSP-1, α-SMA, procollagen I and procollagen III in DOCA group were significantly higher than those in CON group. In addition, in a specific group, the PDGFRβ mRNA expression was higher than the PDGFRα mRNA expression. In DOCA+IMA group, the mRNA expressions of PDGFRβ, FSP-1, α-SMA, procollagen I and procollagen III were markedly reduced when compared with DOCA group. 3) At 14 days, the protein expressions of PDGFRα and PDGFRβ in DOCA group were significantly higher than those in CON group. The PDGFRα protein expression in DOCA+IMA group was markedly lower than that in DOCA group. At days 28, the protein expressions of PDGFRα and PDGFRβ in DOCA group were significantly increased when compared with CON group. The protein expressions of PDGFRα and PDGFRβ in DOCA+IMA group were significantly lower than those in DOCA group. At day 28, the cardiac interstitium mainly contained vimentin positive fibroblasts, and α-SMA positive cells were less identified in CON group. In DOCA group, α-SMA positive fibroblasts (spindle-shaped) increased significantly, but the myofibroblasts reduced significantly in DOCA+IMA group when compared with DOCA group. 4) PDGFRα protein expression was observed in fibroblasts and myofibroblasts, but not in VSMCs. PDGFRβ protein expression was noted in not only fibroblasts and myofibroblasts but also VSMCs. Thus, During myocardial fibrosis of DOCA induced salt-sensitive hypertensive rats, PDGFRα acts at early stage, but PDGFRβ functions in the whole process. PDGFRα and PDGFRβ expressions increase in fibroblasts and myofibroblasts, suggesting that PDGF/PDGFR signaling pathway is involved in the myocardial fibrosis via stimulating fibroblasts to proliferate and transform into myofibroblasts.     

Aldosterone and TGF-β1 synergistically increase PAI-1 expression in hepatic stellate cells of rats

Objective: Aldosterone is related to the fibrosis of several organs, but the specific mechanism underlying the aldosterone induced hepatic fibrosis is still unclear. Methods: Separation, culture and identification of primary hepatic stellate cells (HSCs): The fluids and digestives used in the present study were able to completely remove blood cells, digest hepatocytes and matrix, and effectively separate HSCs. The in situ perfusion was performed at 2 steps: in situ perfusion with pre-perfusion fluid and ex vivo perfusion with enzyme-containing perfusion fluid. Influence of Ald on PAI-1 and Smad expressions in HSCs: cells were divided into control group, Ald group (10^-6 M), spironolactone (SPI) group and Ald+SPI group, and the mRNA and protein expressions of PAI-1 and Smad were detected. Ald induced type I collagen expression in HSCs: Immunohistochemistry was performed to detect type I collagen expression in the supernatant of control group, Ald group (10^-6 M), TGF-β₁ group, and Ald+TGF-β₁ group. Influence of Ald and TGF-β₁ on PAI-1 expression in HSCs: cells were divided into control group, Ald group (10^-6 M), TGF-β₁ group, and Ald+TGF-β₁ group, and the mRNA and protein expressions of PAI-1 were determined by RT-PCR and Western blot assay, respectively. Synergistic effect of Ald and TGF-β₁ on PAI-1 expression in HSCs: cells were divided into control group, Ald group (10^-6), TGF-β₁ group, Ald (10^-6 M)+TGF-β₁ group, Ald (10^-7 M)+TGF-β₁ group and Ald (10^-8 M)+TGF-β₁ group, and the mRNA and protein expressions of PAI-1 were detected by RT-PCR and Western blot assay, respectively. Results: The survival rate, purity, markers and activation of HSCs were determined after separation. Influence of Ald on PAI-1 expression in HSCs: PAI-1 expression increased in HSCs of Ald group, SPI group and Ald+API group, and the PAI-1 expression in Ald group and Ald+SPI group was significantly higher than in control group (P<0.01). Influence of Ald on Smad expression in HSCs: Smad expression in Ald group, TGF-β₁ group and ALD+TGF-β₁ group was markedly higher than in control group (P<0.05). Smad expression in ALD+TGF-β₁ group increased significantly when compared with Ald group (P<0.01). Ald induced type I collagen expression in HSCs: type I collagen expression in Ald group, TGF-β₁ group and ALd+TGF-β₁ group was dramatically higher than in control group (P<0.05), and it in ALd+TGF-β₁ group was also significantly different from that in Ald group and TGF-β₁ group (P<0.01). Synergistic effects of Ald and TGF-β₁ on PAI expression in HSCs: PAI-1 expression in treated cells was markedly higher than in control group (P<0.01). PAI-1 expression in 10^-6 M Ald+5 ng/ml TGF-β₁ group increased dramatically as compared to Ald group and TGF-β₁ group (P<0.01), but the increased PAI-1 expression reduced after SPI treatment. Ald at different concentrations exerts synergistic effect with TGF-β₁ to increase PAI-1 expression in HSCs: PAI-1 expression in HSCs after different treatments increased markedly as compared to control group (P<0.01). Significant difference in PAI-1 expression was observed in 10^-6 M Ald+50 pg/ml TGF-β₁ group and 10^-6 M Ald group (P<0.01), PAI-1 expression in 10^-7 M Ald+50 pg/ml TGF-β₁ group was significantly higher than in 50 pg/ml TGF-β₁ group (P<0.01), but the PAI-1 expression in 10^-7 M Ald+50 pg/ml TGF-β₁ group was similar to that in 10^-6 M Ald group (P>0.05). Conclusion: Aldosterone is able to activate HSCs and increase PAI-1 expression during hepatic fibrosis, which may be inhibited by spironolactone. Aldosterone and TGF-β₁ may synergistically act on HSCs to increase PAI-1 expression as compared to treatment with aldosterone or TGF-β₁ alone. Aldosterone or TGF-β₁ alone may slightly increase PAI-1 expression in HSCs, which can be inhibited by spironolactone.     

Effects of 18α-glycyrrhizin on TGF-β1/Smad signaling pathway in rats with carbon tetrachloride-induced liver fibrosis

Background: Glycyrrhizin has various pharmacological effects including hepato-protection. This study aimed to investigate the potential mechanism underlying the protective effects of 18α-glycyrrhizin (18α-GL) in rats with carbon tetrachloride (CCl₄) induced liver fibrosis. Methods: Male Sprague-Dawley (SD) rats were randomly divided into control group, fibrosis group, 25 mg/kg 18α-GL group and 12.5 mg/kg 18α-GL group. Rats in experimental groups were subcutaneously injected with 40% CCl₄ twice weekly for 8 weeks. Immunohistochemical examination was carried out to detect the protein expressions of collagen I, collagen III, TGF-β1, p-Smad2, p-Smad3, Smad 7 and SP-1, in the liver, and the mRNA and protein expressions of these genes were determined in the liver by real time PCR and Western blot assay, respectively. Results: 18α-GL ameliorated histological changes and significantly suppressed collagen deposition. 18α-GL significantly decreased the mRNA expressions of TGF-β1, Smad2, Smad3 and SP-1 in the liver. Immunohistochemical staining revealed that TGF-β1, p-Smad2, p-Smad3 and SP-1 expressions reduced following 18α-GL therapy. Western blot assay showed p-Smad2, p-Smad3, smad2 and smad3 expressions decreased after 18α-GL treatment. The mRNA and protein expression of Smad7 remained unchanged. Conclusion: 18α-GL is able to attenuate CCl₄ induced liver fibrosis in rat.     

Implication of RAS in Postnatal Cardiac Remodeling,Fibrosis and Dysfunction Induced by Fetal Undernutrition

Fetal undernutrition is a risk factor for cardiovascular diseases. Male offspring from rats exposed to undernutrition during gestation (MUN) exhibit oxidative stress during perinatal life and develop cardiac dysfunction in ageing. Angiotensin-II is implicated in oxidative stress-mediated cardiovascular fibrosis and remodeling, and lactation is a key developmental window. We aimed to assess if alterations in RAS during lactation participate in cardiac dysfunction associated with fetal undernutrition. Control dams received food ad libitum, and MUN had 50% nutrient restriction during the second half of gestation. Both dams were fed ad libitum during lactation, and male offspring were studied at weaning. We assessed: ventricular structure and function (echocardiography); blood pressure (intra-arterially, anesthetized rats); collagen content and intramyocardial artery structure (Sirius red, Masson Trichromic); myocardial and intramyocardial artery RAS receptors (immunohistochemistry); plasma angiotensin-II (ELISA) and TGF-β1 protein expression (Western Blot). Compared to Control, MUN offspring exhibited significantly higher plasma Angiotensin-II and a larger left ventricular mass, as well as larger intramyocardial artery media/lumen, interstitial collagen and perivascular collagen. In MUN hearts, TGF-β1 tended to be higher, and the end-diastolic diameter and E/A ratio were significantly lower with no differences in ejection fraction or blood pressure. In the myocardium, no differences between groups were detected in AT1, AT2 or Mas receptors, with MrgD being significantly lower in the MUN group. In intramyocardial arteries from MUN rats, AT1 and Mas receptors were significantly elevated, while AT2 and MrgD were lower compared to Control. Conclusions. In rats exposed to fetal undernutrition, RAS disbalance and associated cardiac remodeling during lactation may set the basis for later heart dysfunction.     

Protective effects of calcitriol on diabetic nephropathy are mediated by down regulation of TGF-β1 and CIP4 in diabetic nephropathy rat

Objective: To explore the protective effects of calcitriol on diabetic nephropathy by modulating the expressions of transforming growth factor-beta 1 (TGF-β1) and Cdc42 interacting protein-4 (CIP4). Methods: Streptozotocin-induced diabetic nephropathy rats (n=36) were randomly divided into control group (control-H, control-M, control-L) and calcitriol group (calcitriol-H, calcitriol-M, calcitriol-L). The expression of TGF-β1 gradually decreased in control-H, control-M and control-L subgroups by injection of different virus vectors. Peanut oil and calcitriol were given to control and calcitriol group, respectively. The expressions of TGF-β1 and CIP4 in kidney, the pathology, and the renal function and lipid profiles were compared between control and calcitriol treatment groups. Results: In the control group, the higher level of TGF-β1 was associated with more severe glomerular pathology (P<0.05). There is a positive correlation between the expression of CIP4 and TGF-β1. Control-H subgroup had significant more severe kidney disease, higher levels of cholesterol, triglyceride, blood glucose, blood urea nitrogen (BUN) and creatinine (Cr) than control-M and control-L subgroups. After calcitriol treatment, the expression of TGF-β1 and CIP4 were significantly decreased compared to the corresponding control subgroups (all P<0.05). Renal fibrosis and pathological changes were markedly improved. The levels of cholesterol, triglyceride, blood glucose, BUN and Cr were significantly reduced (P<0.05). Conclusion: Calcitriol may protect diabetic nephropathy from fibrosis via inhibition of TGF-β1 and CIP4.     

Possible involvement of TGF-β/periostin in fibrosis of right atrial appendages in patients with atrial fibrillation

Atrial fibrosis contributes to development and recurrence of atrial fibrillation (AF). TGF-β and periostin have been reported to be involved in fibrogenesis. Here we investigated the role of TGF-β and periostin in atrial fibrosis of AF and in the recurrence of AF after surgery ablation. Western blot, Masson staining, immunohistochemistry and colorimetry were performed to detect the degree of atrial fibrosis and the expression of TGF-β, periostin and collagens in 70 biopsies of right atrial appendage (RAA) obtained in this study. Then the patients who received surgical ablation were followed up for about one year. The results showed an increasing gradient of atrial expression of TGF-β, periostin and collagens paralleled by a higher level of atrial fibrosis in control, SR and AF groups. The expression of TGF-β and periostin was significantly correlated with fibrotic markers. In addition, LAD and the expression of TGF-β were larger or higher in recurrence group than that in nonrecurrence group after surgery ablation. The results suggest that upregulated expression of TGF-β and periostin in RAAs is correlated with the degree of atrial fibrosis in patients with AF.     

Effect of losartan with folic acid on plasma homocysteine and vascular ultrastructural changes in spontaneously hypertensive rats

Elevated homocysteine (Hcy) is a high risk factor of hypertension due to its function in endothelial dysfunction. Its level in the blood is strongly influenced by folic acid. In order to investigate the effects of losartan with folic acid on plasma level of Hcy and vascular ultrastructural changes, thirty spontaneously hypertensive rats (SHR) involved and randomly divided into three groups (n=10): SHR-C group (control), SHR-L group (losartan 25 mg·kg^-1·d^-1), SHR-L+Y group (losartan 25 mg·kg^-1·d^-1 + folic acid 0.4 mg·kg^-1·d^-1). Another 10 Wistar Rats involved as WKY-C group for normal control. The level of plasma Hcy was measured dynamically by LS-MS, the vascular ultrastructural changes were analyzed by light and electron microscopy. Moreover, the thickness and area of aorta was measured. The results showed the Hcy levels in four groups were WKY-C 7.49 ± 1.95 μmol/L; SHR-C 8.45 ± 1.90 μmol/L; SHR-L 8.28 ± 2.11 μmol/L; SHR-L+Y 7.53 ± 2.02 μmol/L at 80 days. There was no significant change for plasma Hcy (P>0.05). The morphological change showed the subendothelial space didn’t increased significantly, the endothelial cells have a more smooth and intact cellular membrane in SHR-L+Y group. In conclusion, Losartan combined with folic acid could improve arterial endothelial structure in SHR which has no significant correlation with plasma Hcy.     

Myrtol ameliorates cartilage lesions in an osteoarthritis rat model

Purpose: The aim of this study is to evaluate the effects of myrtol standardized on cartilage lesions in osteoarthritis (OA) rats. Methods: Fifty-six healthy Sprague-Dawley rats were randomly divided into sham group (13 rats) and OA model group (43 rats) with interior meniscus excision. Then serum estradiol (E₂) and glycosaminoglycan (GAG) content in cartilage tissue were measured by radioimmunoassay and toluidine blue staining, respectively. After that, the model rats were randomly divided into low dose myrtol (LDM) group, middle dose myrtol (MDM) group and high dose myrtol (HDM) group (10 rats in each group) with treatment of 450 mg/kg, 300 mg/kg and 150 mg/kg myrtol, respectively. Then, Mankin scores were used to evaluate lesion extent of knee joint cartilage. Expression of tumor necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1), interleukin (IL)-6, Bax and Bcl-2 were investigated using PCR gel electrophoresis method. Results: Mankin cores were lower in sham group and myrtol group than in model group. There were statistically significant differences (P < 0.01) between sham group and model group in expression of TNF-α, TGF-β1, IL-6, Bax and Bcl-2 in the cartilage tissue. Myrtol significantly reduced the expression of TNF-α, IL-6 and Bax, and increased the expression of TGF-β1 and Bcl-2 in myrtol group, comparing with those in model group (P < 0.01). Conclusions: Myrtol could down-regulate the expression of TNF-α, IL-6 and Bax, and up-regulate the expression of TGF-β1 and Bcl-2. Myrtol standardized is a promising drug to ameliorate knee cartilage lesions in the OA rat model.     

Endotoxin-Induced Endothelial Fibrosis Is Dependent on Expression of Transforming Growth Factors β1 and β2

During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases.     

miR-144 regulates transforming growth factor-β1 iduced hepatic stellate cell activation in human fibrotic liver

Objectives: Activation of hepatic stellate cells (HSCs) into collagen producing myofibroblasts is critical for pathogenesis of liver fibrosis. Transforming growth factor-β1 (TGF-β1) is one of the main profibrogenic mediators for HSC transdifferentiation. Recent studies have shown effect of microRNAs (miRNAs) on regulating TGF-β1-induced HSC activation during liver fibrosis. Here, we aimed to explore the roles of miR-144 and miR-200c in human liver fibrosis. Methods: Expression of TGF-β1 was detected in 42 fibrotic and 18 normal human liver tissues by quantitative real time polymerase chain reaction (qRT-PCR) and immunohistochemistry, and its correlation with α-smooth muscle actin (α-SMA) was calculated. miR-144 and miR-200c expression level in fibrotic liver tissues were also detected by qRT-PCR. The correlation of TGF-β1 expression with miR-200c and miR-144 in the fibrotic liver was analyzed. Results: The results showed that TGF-β1 expression was much higher in fibrotic liver than that in normal liver tissues (P<0.05). TGF-β1 protein high expressing liver fibrosis showed α-SMA positive cells in the liver parenchyma indicating activated HSCs. Expression of TGF-β1 in fibrotic liver was significantly correlated with α-SMA expression (R=0.633, P<0.001). Furthermore, miR-144 was less expressed in liver fibrosis (P<0.05) and was significantly correlated with expression of TGF-β1 in fibrotic liver tissues (R=-0.442, P<0.01). However, miR-200c did not show significant difference between normal and fibrotic liver (P=0.48) and correlation with TGF-β1 expression (R=0.106, P=0.51). Conclusion: All the results indicate that miR-144 can be a novel regulator of TGF-β1-induced HSC activation during liver fibrosis.     

BMP-7 attenuates liver fibrosis via regulation of epidermal growth factor receptor

The aim of this study was to elucidate the effect of bone morphogenetic protein-7 (BMP-7) on liver fibrosis induced by carbon tetrachloride (CCl4) in vivo and on the hepatic stellate cells (HSC) activation in vitro. In vivo, thirty male ICR mice were randomly allocated to three groups, the control group (n = 6), the CCl4 group (n = 18) and the BMP-7+CCl4 group (n = 6). The model of liver fibrosis was induced by intraperitoneal injection with CCl4 three times per week lasting for 12 weeks in CCl4 group and the BMP-7+CCl4 group. After 8 weeks injection with CCl4, mice were intraperitoneal injected with human recombinant BMP-7 in BMP-7+CCl4 group. Meanwhile, mice in the CCl4 group were only intraperitoneal injection with equal amount of saline. The degree of liver fibrosis was assessed by HE and Masson’s staining. PCR and western blot were used to detect mRNA and protein levels. In BMP-7+CCl4 group, serum levels of alanine aminotransferase (ALT) and aminotransferase (AST) were decreased and serum albumin (Alb) was increased. Meanwhile, the expressions of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) were down-regulated by BMP-7 intervention as compared to the CCl4 group (P < 0.05). Furthermore, BMP-7 also suppressed the expression of epidermal growth factor receptor (EGFR) and phosphorylated-epidermal growth factor receptor (pEGFR). HE and Masson stain showed that liver damage was alleviated in BMP-7+CCl4 group. In vitro study, expression of EGFR, TGF-β1 and α-SMA were down regulated by BMP-7 dose-dependently, indicating it might effect on suppression of HSC activation. Therefore, our data indicate BMP-7 was capable of inhibiting liver fibrosis and suppressing HSCs activation, and these effects might rely on its crosstalk with EGFR and TGF-β1. We suggest that BMP-7 may be a potential reagentfor the prevention and treatment of liver fibrosis.     

Activation of AMP-Activated Protein Kinase Prevents TGF-β1–Induced Epithelial-Mesenchymal Transition and Myofibroblast Activation

Transforming growth factor (TGF)-β contributes to tubulointerstitial fibrosis. We investigated the mechanism by which TGF-β exerts its profibrotic effects and specifically the role of AMP-activated protein kinase (AMPK) in kidney tubular epithelial cells and interstitial fibroblasts. In proximal tubular epithelial cells, TGF-β1 treatment causes a decrease in AMPK phosphorylation and activation together with increased fibronectin and α-smooth muscle actin expression and decreased in E-cadherin. TGF-β1 causes similar changes in interstitial fibroblasts. Activation of AMPK with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside, metformin, or overexpression of constitutively active AMPK markedly attenuated TGF-β1 functions. Conversely, inhibition of AMPK with adenine 9-β-d-arabinofuranoside or siRNA-mediated knockdown of AMPK (official name PRKAA1) mimicked the effect of TGF-β1 and enhanced basal and TGF-β1–induced phenotypic changes. Importantly, we found that tuberin contributed to the protective effects of AMPK and that TGF-β1 promoted cell injury by blocking AMPK-mediated tuberin phosphorylation and activation. In the kidney cortex of TGF-β transgenic mice, the significant decrease in AMPK phosphorylation and tuberin phosphorylation on its AMPK-dependent activating site was associated with an increase in mesenchymal markers and a decrease in E-cadherin. Collectively, the data indicate that TGF-β exerts its profibrotic action in vitro and in vivo via inactivation of AMPK. AMPK and tuberin activation prevent tubulointerstitial injury induced by TGF-β. Activators of AMPK provide potential therapeutic strategy to prevent kidney fibrosis and progressive kidney disease.Tubulointerstitial fibrosis is a prominent pathologic feature of progressive renal disease that culminates in loss of renal function. Inflammatory and metabolic insults result in kidney fibrosis in which transforming growth factor (TGF)-β plays a prominent role. Tubular epithelial cells and interstitial fibroblast contribute to this process by secreting and remodeling the extracellular matrix. In progressive fibrotic renal disease, TGF-β causes proximal tubular epithelial cells (PTECs) to acquire mesenchymal cell characteristics sometimes referred to as epithelial-mesenchymal transition (EMT).¹ In the presence of TGF-β, interstitial fibroblasts are also activated, differentiate to myofibroblasts, and contribute to the accumulation of extracellular matrix proteins. Accumulation of matrix proteins progressively destroys the normal kidney tissue architecture and disrupts blood flow and nephron function.² The increase in TGF-β1 levels is causally linked to the activation of profibrotic signaling pathways initiated by angiotensin, glucose, and oxidative stress.³ There is substantial evidence supporting a role for AMP-activated protein kinase (AMPK) in multiple diseases, including diabetes mellitus,^4,5 metabolic syndrome,^6–9 and cancer.^10,11 However, the role of AMPK in renal disease is underexplored. AMPK is a phylogenetically conserved serine/threonine kinase that regulates diverse cellular functions.¹² AMPK is heterotrimeric complex comprising a catalytic α (α1, α2) subunit and two regulatory subunits β (β1, β2) and γ (γ1, γ2, γ3). The activity and subunit composition of AMPK are expressed in a cell- and tissue-specific manner, with the α₁ and α₂ subunits expressed in the kidney, including tubular epithelial cells and glomerular cells.^13,14 AMPK activation leads to its phosphorylation at Thr172 in the catalytic domain of the α-subunit. AMPK can also be activated independent of changes in the AMP/ATP ratio.^15–17 On activation, AMPK turns on ATP-generating catabolic pathways and turns off ATP-consuming anabolic pathways.We investigated the role of AMPK in mediating the effect of TGF-β1 in human and murine PTECs, rat kidney interstitial fibroblast cells, and TGF-β1 transgenic mouse model. We provide strong evidence that TGF-β induces EMT phenotype through inactivation of AMPK and that activation of AMPK prevents the effects of TGF-β.     

Rutin ameliorates renal fibrosis and proteinuria in 5/6-nephrectomized rats by anti-oxidation and inhibiting activation of TGFβ1-smad signaling

Objectives: Rutin, a polyphenolic flavonoid, was reported to have beneficial effect on drug induced nephropathy. The present study aimed to introduce 5/6 nephrectomized rat model to further evaluate its renal protective effect. Methods: Adult Wistar rats were induced to develop chronic renal failure through 5/6 nephrectomy (5/6 Nx). After that, animals were treated orally with saline, rutin at 15 and 45 mg/kg, and losartan (10 mg/kg) daily for 20 weeks; sham-operated animals were also involved as control. After treatment for 8 and 20 weeks, blood and urine samples were collected for biochemical examination; all the kidney remnants were collected for histological examination. The protein levels of TGF-β1, smad2 and phosphorylated-smad2 (p-smad2) in kidney were measured. Immunohistochemistry was used to analyze the expression of TGF-β1, fibronectin and collagen IV in kidney tissues. Results: Results suggested that rutin could reduce the proteinurea, blood urine nitrogen and blood creatinine in 5/6 Nx animals significantly, as well as oxidation stress in the kidney. By histological examination, rutin administration alleviated glomerular sclerosis scores and tubulointerstitial injuries in a dose-dependent manner (P<0.01). Immunohistochemistry also suggested rutin could reduce the expression of TGF-β1, fibronectin and collagen IV in kidney tissues. By western blot, we found the rutin could reduce the TGF-β1, p-smad2 expression in the kidney tissues of rats. Conclusions: This study suggests that the rutin can improve renal function in 5/6 Nx rats effectively. Its effect may be due to its anti-oxidation and inhibiting TGFβ1-Smad signaling.     

Protective roles of pulmonary rehabilitation mixture in experimental pulmonary fibrosis in vitro and in vivo

Abnormal high mobility group protein B1 (HMGB1) activation is involved in the pathogenesis of pulmonary fibrosis. Pulmonary rehabilitation mixture (PRM), which combines extracts from eight traditional Chinese medicines, has very good lung protection in clinical use. However, it is not known if PRM has anti-fibrotic activity. In this study, we investigated the effects of PRM on transforming growth factor-β1 (TGF-β1)-mediated and bleomycin (BLM)-induced pulmonary fibrosis in vitro and in vivo. The effects of PRM on TGF-β1-mediated epithelial-mesenchymal transition (EMT) in A549 cells, on the proliferation of human lung fibroblasts (HLF-1) in vitro, and on BLM-induced pulmonary fibrosis in vivo were investigated. PRM treatment resulted in a reduction of EMT in A549 cells that was associated with attenuating an increase of vimentin and a decrease of E-cadherin. PRM inhibited the proliferation of HLF-1 at an IC₅₀ of 0.51 µg/mL. PRM ameliorated BLM-induced pulmonary fibrosis in rats, with reduction of histopathological scores and collagen deposition, and a decrease in α-smooth muscle actin (α-SMA) and HMGB1 expression. An increase in receptor for advanced glycation end-product (RAGE) expression was found in BLM-instilled lungs. PRM significantly decreased EMT and prevented pulmonary fibrosis through decreasing HMGB1 and regulating RAGE in vitro and in vivo. PRM inhibited TGF-β1-induced EMT via decreased HMGB1 and vimentin and increased RAGE and E-cadherin levels. In summary, PRM prevented experimental pulmonary fibrosis by modulating the HMGB1/RAGE pathway.