LRG1 inhibits hepatic macrophage activation by enhancing TGF-β1 signaling to alleviate MAFLD in mice / 南方医科大学学报

OBJECTIVE@#To explore the effect of leucine-rich α-2-glycoprotein (LRG1) derived from hepatocytes on activation of hepatic M1 Kupffer cells.@*METHODS@#A metabolic dysfunction-associated fatty liver disease (MAFLD) model was established in BALB/c mice by high-fat diet (HFD) feeding for 16 weeks. Oleic acid was used to induce steatosis in primary cultures of mouse hepatocytes. The mRNA and protein expressions of LRG1 in mouse liver tissues and hepatocytes were detected by real-time PCR and Western blotting. Primary hepatic macrophages were stimulated with the conditioned medium (CM) from steatotic hepatocyte along with LRG1 or transforming growth factor-β1 (TGF-β1), or both for 24 h, and the expression levels of inducible nitric oxide synthase (iNOS) was detected with Western botting, and the mRNA expressions of iNOS, chemokine ligand 1 (CXCL-1) and interleukin-1β (IL-1β) were measured by RT-PCR. The MAFLD mice were injected with LRG1 (n=6), TGF-β1 (n=6), or both (n=6) through the caudal vein, and the live tissues were collected for HE staining and immumohistochemical detection of F4/80 expression; the mRNA expressions of iNOS, CXCL-1 and IL-1β in liver tissues were detected using RT-PCR.@*RESULTS@#The mRNA and protein expression levels of LRG1 were significantly downregulated in the liver tissues of MAFLD mice and steatotic hepatocytes (P < 0.05). Treatment of the hepatic macrophages with CM from steatosis hepatocytes significantly enhanced the mRNA expression levels of iNOS, CXCL-1 and IL-1β, and these changes were significantly inhibited by the combined treatment with TGF-β1 and LRG1 (P < 0.05). In MAFLD mice, injections with either LRG1 or TGF-β1 alone reduced hepatic lipid deposition and intrahepatic macrophage infiltration, and these effects were significantly enhanced by their combined treatment, which also more strongly inhibited the mRNA expression levels of iNOS, CXCL-1 and IL-1β (P < 0.05).@*CONCLUSION@#LRG1 inhibits hepatic macrophage infiltration by enhancing TGF-β1 signaling to alleviate fatty liver inflammation in MAFLD mice.

Role of TGF-β/Smad signaling pathway in diabetic kidney disease and research progress of traditional Chinese medicine intervention / 中国中药杂志

Diabetic kidney disease is an important microvascular complication of diabetes and the leading cause of end-stage renal disease. Its pathological characteristics mainly include epithelial mesenchymal transition(EMT) in glomerulus, podocyte apoptosis and autophagy, and damage of glomerular filtration barrier. Transforming growth factor-β(TGF-β)/Smad signaling pathway is specifically regulated by a variety of mechanisms, and is a classic pathway involved in physiological activities such as apoptosis, proliferation and differentiation. At present, many studies have found that TGF-β/Smad signaling pathway plays a key role in the pathogenesis of diabetic kidney disease. Traditional Chinese medicine has significant advantages in the treatment of diabetic kidney disease for its multi-component, multi-target and multi-pathway characteristics, and some traditional Chinese medicine extracts, traditional Chinese medicines and traditional Chinese medicine compound prescription improve the renal injury of diabetic kidney disease by regulating TGF-β/Smad signaling pathway. This study clarified the mechanism of TGF-β/Smad signaling pathway in diabetic kidney disease by expounding the relationship between the key targets of the pathway and diabetic kidney disease, and summarized the research progress of traditional Chinese medicine in the treatment of diabetic kidney disease by interfering with TGF-β/Smad signaling pathway in recent years, to provide reference for drug research and clinical treatment of diabetic kidney disease in the future.

Effect of mycophenolate mofetil alleviates carbon tetrachloride-induced liver fibrosis in mice / 中南大学学报(医学版)

OBJECTIVES@#Hepatic fibrosis is a serious pathological consequence of chronic liver disease. Mycophenolate mofetil (MMF) is a commonly used immunosuppressant after organ transplant. However, the relationship between MMF and hepatic fibrosis remains unclear. This study aims to explore the effect of MMF on hepatic fibrosis in mice and the potential mechanism.@*METHODS@#A total of 24 mice (male, 8-week old, C57BL/6) were randomly divided into a control group, a MMF group, a carbon tetrachloride (CCl4) group and a CCl4+MMF group (n=6 in each group). After the mice were sacrificed, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected. The liver tissues were taken up for Masson staining and collagen I (COL1) immunohistochemistry. The levels of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) were detected by Western blotting. Finally, the levels of mRNA for TGF-β1, α-SMA, and COL1 were detected using real-time PCR.@*RESULTS@#Compared with the CCl4 group, the ALT and AST levels were lower (both P<0.05), the degree of liver fibrosis was alleviated, and the deposition of COL1 in the liver was significantly decreased (P<0.01) in the CCl4+MMF group. Compared with the CCl4 group, the protein expression levels of TGF-β1 and α-SMA were significantly decreased (both P<0.05) and the relative expression levels of TGF-β1, α-SMA and COL1 mRNA in the liver were significantly decreased (all P<0.05) in the CCl4+MMF.@*CONCLUSIONS@#MMF could reduce CCl4-induced hepatic fibrosis, which might be related to the inhibition of TGF-β1. This study is expected to provide a target for the treatment of hepatic fibrosis.

Inhibitory Effect of Resveratrol on LPS-induced Glomerular Mesangial Cells Proliferation and TGF-β1 Expression via Sphingosine Kinase 1 Pathway / 中国结合医学杂志

OBJECTIVE@#To elucidate the renoprotective effect of resveratrol (RSV) on sphingosine kinase 1 (SphK1) signaling pathway and expression of its downstream molecules including activator protein 1 (AP-1) and transformation growth factor-β1 (TGF-β1) in lipopolysaccharide (LPS)-induced glomerular mesangial cells (GMCs).@*METHODS@#The rat GMCs line (HBZY-1) were cultured and randomly divided into 5 groups, including control, LPS (100 ng/mL), and 5, 10, 20 µmol/L RSV-treated groups. In addition, SphK1 inhibitor (SK-II) was used as positive control. GMCs were pretreated with RSV for 2 h and treated with LPS for another 24 h. GMCs proliferation was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The proteins expression of SphK1, p-c-Jun and TGF-β1 in GMCs were detected by Western blot, and DNA-binding activity of AP-1 was performed by electrophoretic mobility shift assay (EMSA). The binding activity between RSV and SphK1 protein was detected by AutoDock Vina and visualized by Discovery Studio 2016.@*RESULTS@#LPS could obviously stimulate GMCs proliferation, elevate SphK1, p-c-Jun and TGF-β1 expression levels and increase the DNA-binding activity of AP-1 (P<0.05 or P<0.01), whereas these effects were significantly blocked by RSV pretreatment. It was also suggested that the effect of RSV was similar to SK-II (P>0.05). Moreover, RSV exhibited good binding affinity towards SphK1, with docking scores of -8.1 kcal/moL and formed hydrogen bonds with ASP-178 and LEU-268 in SphK1.@*CONCLUSION@#RSV inhibited LPS-induced GMCs proliferation and TGF-β1 expression, which may be independent of its hypoglycemic effect on preventing the development of mesangial cell fibrosis and closely related to the direct inhibition of SphK1 pathway.

Amygdalin Ameliorates Liver Fibrosis through Inhibiting Activation of TGF-β/Smad Signaling / 中国结合医学杂志

OBJECTIVE@#To observe the effect of amygdalin on liver fibrosis in a liver fibrosis mouse model, and the underlying mechanisms were partly dissected in vivo and in vitro.@*METHODS@#Thirty-two male mice were randomly divided into 4 groups, including control, model, low- and high-dose amygdalin-treated groups, 8 mice in each group. Except the control group, mice in the other groups were injected intraperitoneally with 10% carbon tetrachloride (CCl4)-olive oil solution 3 times a week for 6 weeks to induce liver fibrosis. At the first 3 weeks, amygdalin (1.35 and 2.7 mg/kg body weight) were administered by gavage once a day. Mice in the control group received equal quantities of subcutaneous olive oil and intragastric water from the fourth week. At the end of 6 weeks, liver tissue samples were harvested to detect the content of hydroxyproline (Hyp). Hematoxylin and eosin and Sirius red staining were used to observe the inflammation and fibrosis of liver tissue. The expressions of collagen I (Col-I), alpha-smooth muscle actin (α-SMA), CD31 and transforming growth factor β (TGF-β)/Smad signaling pathway were observed by immunohistochemistry, quantitative real-time polymerase chain reaction and Western blot, respectively. The activation models of hepatic stellate cells, JS-1 and LX-2 cells induced by TGF-β1 were used in vitro with or without different concentrations of amygdalin (0.1, 1, 10 µmol/L). LSECs. The effect of different concentrations of amygdalin on the expressions of liver sinusoidal endothelial cells (LSECs) dedifferentiation markers CD31 and CD44 were observed.@*RESULTS@#High-dose of amygdalin significantly reduced the Hyp content and percentage of collagen positive area, and decreased the mRNA and protein expressions of Col-I, α-SMA, CD31 and p-Smad2/3 in liver tissues of mice compared to the model group (P<0.01). Amygdalin down-regulated the expressions of Col-I and α-SMA in JS-1 and LX-2 cells, and TGFβ R1, TGFβ R2 and p-Smad2/3 in LX-2 cells compared to the model group (P<0.05 or P<0.01). Moreover, 1 and 10 µmol/L amygdalin inhibited the mRNA and protein expressions of CD31 in LSECs and increased CD44 expression compared to the model group (P<0.05 or P<0.01).@*CONCLUSIONS@#Amygdalin can dramatically alleviate liver fibrosis induced by CCl4 in mice and inhibit TGF-β/Smad signaling pathway, consequently suppressing HSCs activation and LSECs dedifferentiation to improve angiogenesis.

1, 25-(OH)2-VitD3 attenuates renal tubulointerstitial fibrosis in diabetic kidney disease by inhibiting Snail1-SMAD3/SMAD4 complex formation / 细胞与分子免疫学杂志

Objective To investigate the effect of 1, 25-(OH)2-VitD3 (VitD3) on renal tubuleinterstitial fibrosis in diabetic kidney disease. Methods NRK-52E renal tubular epithelial cells were divided into control group (5.5 mmol/L glucose medium treatment), high glucose group (25 mmol/L glucose medium treatment) and high glucose with added VitD3 group (25 mmol/L glucose medium combined with 10-8 mmol/L VitD3). The mRNA and protein expression of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in NRK-52E cells were detected by real-time quantitative PCR and Western blot analysis respectively. The expression and localization of Snail1, SMAD3 and SMAD4 were detected by immunofluorescence cytochemical staining. The binding of Snail1 with SMAD3/SMAD4 complex to the promoter of Coxsackie-adenovirus receptor (CAR) was detected by chromatin immunoprecipitation. The interaction among Snail1, SMAD3/SMAD4 and E-cadherin were detected by luciferase assay. Small interfering RNA (siRNA) was used to inhibit the expression of Snail1 and SMAD4, and the expression of mRNA of E-cadherin was detected by real-time quantitative PCR. SD rats were randomly divided into control group, DKD group and VitD3-treated group. DKD model was established by injection of streptozotocin (STZ) in DKD group and VitD3-treated group. After DKD modeling, VitD3-treated group was given VitD3 (60 ng/kg) intragastric administration. Control group and DKD group were given normal saline intragastric administration. In the DKD group and VitD3-treated group, insulin (1-2 U/kg) was injected subcutaneously to control blood glucose for 8 weeks. The mRNA and protein levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissues were detected by real-time quantitative PCR and Western blot analysis respectively. Immunohistochemistry was used to detect the expression and localization of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in renal tissue. Results Compared with the control group, the mRNA and protein expressions of Snail1, SMAD3, SMAD4 and α-SMA in NRK-52E cells cultured with high glucose and in DKD renal tissues were up-regulated, while E-cadherin expression was down-regulated. After the intervention of VitD3, the expression levels of Snail1, SMAD3, SMAD4, α-SMA and E-cadherin in the DKD model improved to be close to those in the control group. Chromatin immunoprecipitation showed that Snail1 and SMAD3/SMAD4 bound to CAR promoter IV, while VitD3 prevented Snail1 and SMAD3/SMAD4 from binding to CAR promoter IV. Luciferase assay confirmed the interaction among Snail1, SMAD3/SMAD4 and E-cadherin. After the mRNA of Snail1 and SMAD4 was inhibited by siRNA, the expression of E-cadherin induced by high glucose was up-regulated. Conclusion VitD3 could inhibit the formation of Snail1-SMAD3/SMAD4 complex and alleviate the renal tubulointerstitial fibrosis in DKD.

Effect of Isodon ternifolius-medicated serum on hepatic stellate cells based on TLR4/NF-κB/NLRP3 signaling pathway / 中国中药杂志

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-β1(TGF-β1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-β1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.

Shenmai Injection Improves Hypertensive Heart Failure by Inhibiting Myocardial Fibrosis via TGF-β 1/Smad Pathway Regulation / 中国结合医学杂志

OBJECTIVE@#To study effects of Shenmai Injection on hypertensive heart failure and its mechanism for inhibiting myocardial fibrosis.@*METHODS@#Salt-sensitive (Dahl/SS) rats were fed with normal diet (0.3% NaCl) and the high-salt diet (8% NaCl) to observe the changes in blood pressure and heart function, as the control group and the model group. Salt-insensitive rats (SS-13BN) were fed with the high-salt diet (8% NaCl) as the negative control group. After modeling, the model rats were randomly divided into heart failure (HF) group, Shenmai Injection (SMI) group and pirfenidone (PFD) group by a random number table, with 6 rats in each group. They were given sterilized water, SMI and pirfenidone, respectively. Blood pressure, cardiac function, fibrosis and related molecular expression were detected by sphygmomanometer, echocardiogram, enzyme linked immunosorbent assay (ELISA), hematoxylin-eosin staining, Masson staining, immunofluorescence and qPCR analysis.@*RESULTS@#After high-salt feeding, compared with the control and negative control group, in the model group the blood pressure increased significantly, the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were significantly reduced, and the serum NT-proBNP concentration increased significantly (all P<0.05); furthermore, the arrangement of myocardial cells was disordered, the edema was severe, and the degree of myocardial fibrosis was also significantly increased (P<0.05); the protein and mRNA expressions of collagen type I (Col I) were up-regulated (P<0.05), and the mRNA expressions of transforming growth factor β 1 (TGF- β 1), Smad2 and Smad3 were significantly up-regulated (P<0.05). Compared with HF group, after intervention of Shenmai Injection, LVEF and LVFS increased, myocardial morphology was improved, collagen volume fraction decreased significantly (P<0.05), and the mRNA expressions of Col I, TGF- β 1, Smad2 and Smad3, as well as Col I protein expression, were all significantly down-regulated (all P<0.05).@*CONCLUSION@#Myocardial fibrosis is the main pathological manifestation of hypertensive heart failure, and Shenmai Injection could inhibit myocardial fibrosis and effectively improve heart failure by regulating TGF-β 1/Smad signaling pathway.

Effect of pirfenidone on paraquat-induced pulmonary fibrosis in rats / 中华劳动卫生职业病杂志

Objective: To construct paraquat (PQ) poisoning rat model and to explore the effect of pirfenidone (PFD) on PQ-induced pulmonary fibrosis. Methods: In April 2017, male 6-8 week-old Wistar rats were selected, and PQ was administered intraperitoneally at one time. PFD was administered by gavage 2 hours after poisoning. The daily gavage doses were 100, 200 and 300 mg/kg, and the rats were divided into physiological saline group, PQ group, PQ+PFD 100 group, PQ+PFD 200 group, PQ+PFD 300 group, with 10 rats in each group at each observation time point. The pathological changes of lung tissue at different time points (the 1st, 3rd, 7th, 14th, 28th, 42nd and 56th days) after poisoning and the effect of PFD intervention with different dose on PQ-induced pulmonary fibrosis were observed. Pathological evaluation of lung tissue was performed by Ashcroft scale method. The PQ+PFD 200 group was selected to further explore the pathological changes of lung tissue, the contents of hydroxyproline and malondialdehyde in lung tissue were determined.And the tumor necrosis factor (TNF) -α, interleukin (IL) -6, transforming growth factor (TGF) -β1, fibroblast growth factor (FGF) -B, platelet-derived growth factor (PDGF) -AB, insulin-like growth factor (IGF) -1 and PQ concentrations in serum and lung tissue were determined. Results: On the 1st to 7th day after PQ exposure, rats developed lung inflammation, which was aggravated on the 7th to 14th day, and pulmonary fibrosis appeared on the 14th to 56th day. Compared with PQ group, the Ashcroft scores of lung fibrosis in PQ+PFD 200 group and PQ+PDF 300 group decreased significantly in 7th and 28th day (P<0.05), while the Ashcroft score of lung fibrosis in PQ+PFD 100 group had no significant difference (P>0.05). After PQ exposure, the content of hydroxyproline in lung tissue increased gradually and reached the peak value on the 28th day. Compared with the PQ group, the contents of hydroxyproline in the PQ+PFD 200 group decreased at the 7th, 14th and 28th day, and the contents of malondialdehyde decreased at the 3rd and 7th day, the differences were statistically significant (P<0.05). The levels of TNF-α, IL-6 in rat serum and lung tissue reached the peak value on the 7th day after PQ exposure, and the levels of TGF-β1, FGF-B and IGF-1 in rat serum and lung tissue reached the peak value on the 14th day after PQ exposure, and the level of PDGF-AB in rat serum and lung tissue reached the peak value on the 28th day after PQ exposure. Compared with PQ group, the level of serum IL-6 in PQ+PFD 200 group decreased significantly on the 7th day, and serum TGF-β1, FGF-B, PDGF-AB and IGF-1 on the 14th and 28th day were decreased significantly (P<0.05). The levels of TNF-α, IL-6 in lung tissue of rats in PQ+PFD 200 group on the 7th day decreased significantly, and the levels of TGF-β1, FGF-B and IGF-1 in lung tissue of rats on the 14th day were significantly decreased, and the level of PDGF-AB in lung tissue of rats on the 28th day were significantly decreased (P<0.05) . Conclusion: PFD partially alleviates the PQ-induced lung inflammation and fibrosis by inhibiting oxidative stress, reducing the levels of pro-inflammatory and pro-fibrotic cytokines in serum and lung tissue, but does not affect the concentrations of PQ in serum and lung tissue.

Effects of Nintedanib associated with Shenfu Injection on paraquat-induced lung injury in rats / 中华劳动卫生职业病杂志

Objective: To study the effects of Nintedanib associated with Shenfu Injection on lung injury induced by paraquat (PQ) intoxication. Methods: In September 2021, a total of 90 SD rats were divided into 5 groups in random, namely control group, PQ poisoning group, Shenfu Injection group, Nintedanib group and associated group, 18 rats in each group. Normal saline was given by gavage route to rats of control group, 20% PQ (80 mg/kg) was administered by gavage route to rats of other four groups. 6 hours after PQ gavage, Shenfu Injection group (12 ml/kg Shenfu Injection), Nintedanib group (60 mg/kg Nintedanib) and associated group (12 ml/kg Shenfu Injection and 60 mg/kg Nintedanib) were administered with medicine once a day. The levels of serum transforming growth factor beta1 (TGF-β1), interleukin-1 beta (IL-1β) were determined at 1, 3 and 7 d, respectively. The pathological changes of lung tissue, the ratio of wet weight and dry weight (W/D) of lung tissue, the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in lung tissue were observed and determined after 7 d. Western blot was used to analyse the expression levels of fibroblast growth factor receptor 1 (FGFR1), platelet derivation growth factor receptor alpha (PDGFRα), vascular endothelial growth factor receptor 2 (VEGFR2) in lung tissue after 7 d. Results: The levels of TGF-β1, IL-1β in all poisoning groups went up first and then went down. The levels of TGF-β1, IL-1β in associated group at 1, 3, 7 d were lower than that of PQ poisoning group, Shenfu Injection group and Nintedanib group at the same point (P<0.05). Pathological changes of lung tissue under the light microscopes showed that the degrees of hemorrhage, effusion and infiltration of inflammatory cells inside the alveolar space of Shenfu Injection group, Nintedanib group and associated group were milder than that of PQ poisoning group, and the midest in associated group. Compared with control group, the W/D of lung tissue was higher, the level of MDA in lung tissue was higher, while the level of SOD was lower, the expressions of FGFR1, PDGFRα and VEGFR2 in lung tissue were higher in PQ poisoning group (P<0.05). Compared with PQ poisoning group, Shenfu Injection group and Nintedanib group, the W/D of lung tissue was lower, the level of MDA in lung tissue was lower, while the level of SOD was higher, the expressions of FGFR1, PDGFRα and VEGFR2 in lung tissue were lower in associated group (P<0.05) . Conclusion: Nintedanib associated with Shenfu Injection can relieve lung injury of rats induced by PQ, which may be related to Nintedanib associated with Shenfu Injection can inhibit the activation of TGF-β1 and the expressions of FGFR1, PDGFRα, VEGFR2 in lung tissue of rats.

Research progress of anti-fibrotic drugs that inhibit epithelial-mesenchymal transition in pulmonary fibrosis / 中华劳动卫生职业病杂志

Pulmonary fibrosis is the end-stage pathological change of lung diseases, which seriously affects the respiratory function of human body. A large number of studies at home and abroad have confirmed that epithelial-mesenchymal transition (EMT) is an important intermediate stage in the development of pulmonary fibrosis. Inhibition of multiple pathways upstream and downstream of EMT, such as the classical Smads pathway and non-Smads pathway of TGF-1 can effectively inhibit the process of EMT and alleviate pulmonary fibrosis. This article will review the main conclusions of the mechanism of action of EMT as a target to improve the pathology of pulmonary fibrosis so far, and provide a theoretical basis and research direction for further research and development of anti-pulmonary fibrosis drugs.

Protective effects of three kinds of borneol on different brain regions in acute cerebral ischemia/reperfusion model rats / 中国中药杂志

This study compared the ameliorating effects of L-borneol, natural borneol, and synthetic borneol on the injury of different brain regions in the rat model of acute phase of cerebral ischemia/reperfusion(I/R) for the first time, which provides a reference for guiding the rational application of borneol in the early treatment of ischemic stroke and has important academic and application values. Healthy specific pathogen-free(SPF)-grade SD male rats were randomly assigned into 13 groups: a sham-operation group, a model group, a Tween model group, a positive drug(nimodipine) group, and high-, medium-, and low-dose(0.2, 0.1, and 0.05 g·kg~(-1), respectively) groups of L-borneol, natural borneol, and synthetic borneol according to body weight. After 3 days of pre-administration, the rat model of I/R was established by suture-occluded method and confirmed by laser speckle imaging. The corresponding agents in different groups were then administered for 1 day. The body temperature was monitored regularly before pre-administration, days 1, 2, and 3 of pre-administration, 2 h after model awakening, and 1 d after model establishment. Neurological function was evaluated based on Zea-Longa score and modified neurological severity score(mNSS) 2 h and next day after awakening. The rats were anesthetized 30 min after the last administration, and blood was collected from the abdominal aorta. Enzyme-linked immunoassay assay(ELISA) was employed to determine the serum levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-4, and transforming growth factor-beta1(TGF-β1). The brain tissues were stained with triphenyltetrazolium chloride(TTC) for the calculation of cerebral infarction rate, and hematoxylin-eosin(HE) staining was used for observing and semi-quantitatively evaluating the pathological damage in different brain regions. Immunohistochemistry was employed to detect the expression of ionized calcium binding adapter molecule 1(IBA1) in microglia. q-PCR was carried out to determine the mRNA levels of iNOS and arginase 1(Arg1), markers of polarization phenotype M1 and M2 in microglia. Compared with the sham-operation group, the model group and the Tween model group showed significantly elevated body temperature, Zea-Longa score, mNSS, and cerebral infarction rate, severely damaged cortex, hippocampus, and striatum, increased serum levels of IL-6 and TNF-α, and decreased serum levels of IL-4 and TGF-β1. The three borneol products had a tendency to reduce the body temperature of rats 1 day after modeling. Synthetic borneol at the doses of 0.2 and 0.05 g·kg~(-1), as well as L-borneol of 0.1 g·kg~(-1), significantly reduced Zea-Longa score and mNSS. The three borneol products at the dose of 0.2 g·kg~(-1) significantly reduced the cerebral infarction rate. L-borneol at the doses of 0.2 and 0.1 g·kg~(-1) and natural borneol at the dose of 0.1 g·kg~(-1) significantly reduced the pathological damage of the cortex. L-borneol and natural borneol at the dose of 0.1 g·kg~(-1) attenuated the pathological damage of hippocampus, and 0.2 g·kg~(-1) L-borneol attenuated the damage of striatum. The 0.2 g·kg~(-1) L-borneol and the three doses of natural borneol and synthetic borneol significantly reduced the serum level of TNF-α, and the 0.1 g·kg~(-1) synthetic borneol reduced the level of IL-6. L-borneol and synthetic borneol at the dose of 0.2 g·kg~(-1) significantly inhibited the activation of cortical microglia, and 0.2 g·kg~(-1) L-borneol up-regulated the expression of Arg1 and down-regulated the expression level of iNOS. In conclusion, the three borneol products may alleviate inflammation to ameliorate the pathological damage of brain regions of rats in the acute phase of I/R by inhibiting the activation of microglia and promoting the polarization of microglia from M1 type to M2 type. The protective effect on brain followed a trend of L-borneol > synthetic borneol > natural borneol. We suggest L-borneol the first choice for the treatment of I/R in the acute phase.

Effect mechanism of acupuncture for anti-asthmatic airway remodeling based on TGF-β1 / Smad3 signaling pathway / 中国针灸

OBJECTIVE@#To observe the effect of acupuncture at "Feishu" (BL 13) + "Dingchuan" (EX-B 1) and "Kongzui" (LU 6) + "Yuji" (LU 10) for the airway remodeling in asthma rats based on the transforming growth factor-β1 (TGF-β1)/ Smad family member 3 (Smad3) signaling pathway; and explore the efficacy difference between the two acupoint combinations.@*METHODS@#Forty SPF male SD rats, aged 4 weeks, were randomly divided into a blank group (n = 10) and a modeling group (n = 30). The ovalbumin (OVA) sensitization method was used to establish asthma model in the modeling group. After successful model preparation, the rats of the modeling group were randomized into a model group, an acupuncture at "Feishu" (BL 13) + "Dingchuan" (EX-B 1) (AAF) group, and acupuncture at "Kongzui" (LU 6)+"Yuji" (LU 10) (AAK) group, with 10 rats in each one. Starting from day 15 of the experiment, 5 min after motivating, acupuncture was applied to "Feishu" (BL 13) + "Dingchuan" (EX-B 1) and "Kongzui" (LU 6)+"Yuji" (LU 10) in the AAF group and the AAK group respectively. The intervention was delivered for 30 min each time, once daily, lasting 3 weeks consecutively. Using lung function detector, the airway resistance (RL) and dynamic compliance (Cdyn) of the lungs were detected. The histomorphology of lung tissues was detected with HE staining and Masson staining, and the mRNA and protein expression of TGF-β1 and Smad3 in lung tissues was detected with the real-time PCR and Western blot methods.@*RESULTS@#Compared with the blank group, RL was increased and Cdyn was decreased in the rats of the model group (P<0.01); and RL was reduced and Cdyn was increased in the AAF group and the AAK group when compared with those in the model group (P<0.01, P<0.05). The rats of the model group had bronchial lumen stenosis, inflammatory cell infiltration, collagen fibre hyperplasia and thickened smooth muscle in the lung tissues when compared with those in the blank group; and in comparison with the model group, all of the above morphological changes were attenuated in the AAF group and the AAK group. Besides, these morphological changes of the lung tissues were more alleviated in the AAF group when compared with those in the AAK group. In comparison with the blank group, the mRNA and protein expression of TGF-β1 and Smad3 of the lung tissues was increased in the model group (P<0.01), and it was reduced in the AAF group and the AAK group when compared with that in the model group (P<0.05, P<0.01). The mRNA expression of TGF-β1 and Smad3 was lower in the AAF group when compared with that in the AAK group (P<0.05).@*CONCLUSION@#Acupuncture at either "Feishu" (BL 13)+"Dingchuan" (EX-B 1) or "Kongzui" (LU 6)+"Yuji" (LU 10) reduces the airway remodeling in the rats with asthma, which may be related to the down-regulation of mRNA and protein expression of TGF-β1 and Smad3. The better efficacy is obtained with acupuncture at "Feishu" (BL 13)+"Dingchuan" (EX-B 1).

Huangqi Decoction, a compound Chinese herbal medicine, inhibits the proliferation and activation of hepatic stellate cells by regulating the long noncoding RNA-C18orf26-1/microRNA-663a/transforming growth factor-β axis / 中西医结合学报

OBJECTIVE@#Huangqi Decoction (HQD), a classical traditional Chinese medicine formula, has been used as a valid treatment for alleviating liver fibrosis; however, the underlying molecular mechanism is still unknown. Although our previous studies showed that microRNA-663a (miR-663a) suppresses the proliferation and activation of hepatic stellate cells (HSCs) and the transforming growth factor-β/small mothers against decapentaplegic (TGF-β/Smad) pathway, whether long noncoding RNAs (lncRNAs) are involved in HSC activation via the miR-663a/TGF-β/Smad signaling pathway has not yet reported. The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis.@*METHODS@#The expression levels of lnc-C18orf26-1, miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction. HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs. The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs. Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs. RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs. Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA. The expression levels of collagen α-2(I) chain (COL1A2), α-smooth muscle actin (α-SMA) and TGF-β/Smad signaling pathway-related proteins were determined using Western blotting.@*RESULTS@#Lnc-C18orf26-1 was upregulated in TGF-β1-activated HSCs and competitively bound to miR-663a. Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation, downregulated TGF-β1-stimulated α-SMA and COL1A2 expression, and inhibited the TGF-β1/Smad signaling pathway. HQD suppressed the proliferation and activation of HSCs. HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs. Further studies showed that HQD inhibited the expression of COL1A2, α-SMA, TGF-β1, TGF-β type I receptor (TGF-βRI) and phosphorylated Smad2 (p-Smad2) in HSCs, and these effects were reversed by miR-663a inhibitor treatment.@*CONCLUSION@#Our study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis. HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-β1/TGF-βRI/p-Smad2 axis.

BMP-4, TGF-β and Smad3 as Modulators of Synovial Fluid Cells Viability / BMP-4, TGF-β e Smad3 como moduladores da viabilidade das células do líquido sinovial

Abstract Objective Our goal was to evaluate the modulation of the synovial fluid cells (SFC) from patients with and without osteoarthritis (OA) by bone morphogenetic protein 4 (BMP-4), Smad-3 and transforming growth factor beta (TGF-β). Methods Synovial fluid was collected from patients submitted to knee arthroscopy or replacement and were centrifuged to isolate cells from the fluid. Cells were cultured for 21 days and characterized as mesenchymal stem cells (MSCs) according to the criteria of the International Society of Cell Therapy. Then, we performed an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (MTT) assay after exposing cells with and without OA to TGF-β, Smad3 and BMP-4 pathway inhibitors and to different concentrations of BMP4. Results Exposure to the TGF-β, Smad3 and BMP-4 inhibitors modifies the mitochondrial activity of the SFCs. The activity of the SFCs is modified by influences of increasing concentrations of BMP4, but there is no difference in cellular activity between patients with and without OA. Conclusion TGF-β, Smad3 and BMP-4 modulate the activity of SFCs from patients with and without knee OA.

Resumo Objetivo Nosso objetivo foi avaliar a modulação das células do líquido sinovial (SFCs, na sigla em inglês) de pacientes com e sem osteoartrite (OA) por proteína morfogenética óssea 4 (BMP-4), Smad3 e transformador do fator de crescimento β (TGF-β). Métodos O do líquido sinovial foi coletado de pacientes submetidos a artroscopia ou artroplastia do joelho, e centrifugados para isolar as células do liquido sinovial. As células foram cultivadas por 21 dias e caracterizadas como células-tronco mesenquimais (MSCs, na sigla em inglês) de acordo com os critérios da International Society of Cell Therapy. Em seguida, realizamos um ensaio de brometo de 3-4,5-dimetil-tiazol-2-il-2,5difeniltetrazólio (MTT) depois de expor células com e sem OA para TGF-β, inibidores de via Smad3 e BMP-4 e para diferentes concentrações de BMP-4. Resultados A exposição aos inibidores TGF-β, Smad3 e BMP-4 modifica a atividade mitocondrial das SFCs. A atividade das SFCs é modificada por influências sobre o aumento das concentrações de BMP-4, mas não há diferença na atividade celular entre pacientes com e sem OA. Conclusão TGF-β, Smad3 e BMP-4 modulam a atividade das SFCs de pacientes com e sem OA do joelho.

TGF-ß1 and CD68 immunoexpression in capsules formed by textured implants with and without mesh coverage: a study on female rats

Purpose: To evaluate fibrosis formation and number of macrophages in capsules formed around textured implants without and with mesh coverage. Methods: Fibrosis was analyzed through transforming growth factor-beta 1 (TGF-ß1) immunomarker expression and the number of macrophages through CD68 percentage of cells in magnified field. Sixty female Wistar rats were distributed into two groups of 30 rats (unmeshed and meshed). Each group was then subdivided into two subgroups for postoperative evaluation after 30 and 90 days. The p value was adjusted by Bonferroni lower than 0.012. Results: No difference was observed in fibrosis between meshed and unmeshed groups (30 days p = 0.436; 90 days p = 0.079) and from 30 to 90 days in the unmeshed group (p = 0.426). The meshed group showed higher fibrosis on the 90th day (p = 0.001). The number of macrophages was similar between groups without and with mesh coverage (30 days p = 0.218; 90 days p = 0.044), and similar between subgroups 30 and 90 days (unmeshed p = 0.085; meshed p = 0.059). Conclusions: In the meshed group, fibrosis formation was higher at 90 days and the mesh-covered implants produced capsules similar to microtextured ones when analyzing macrophages. Due to these characteristics, mesh coating did not seem to significantly affect the local fibrosis formation.

Expression and role of the TRPC family in TGF-β1-induced calcium influx in podocytes / 生理学报

The TRPC family consists of multiple important cationic channels in mammals that participate in a variety of physiological and pathological processes. Our previous studies have shown that transforming growth factor-β1 (TGF-β1) increases the expression of TRPC6 in podocytes, but the roles of other members of the TRPC family in podocytes require further investigation. In this study, we investigated the effect of TGF-β1 on the expression of the TRPC family and the role of the TRPC family in the changes of the intracellular Ca2+ concentration ([Ca2+]i) in podocytes induced by TGF-β1. The model of podocyte injury was established by treatment with TGF-β1 in immortalized glomerular podocytes (MPC5) in vitro. qRT-PCR and Western blot were used to detect the effect of TGF-β1 on the mRNA and protein expression of each TRPC family member. After the expression of each TRPC family member was knocked down by a siRNA-based approach and blocked by SKF96365, respectively, free cytosolic Ca2+ was measured using the fluorescent Ca2+ indicator Fluo-3/AM, and the dynamic change of [Ca2+]i in podocytes was detected by a dynamic high-speed calcium imaging system. The results showed that TGF-β1 increased the protein expression of TRPC1/3/6 in podocytes, but had no effects on the protein expression of TRPC4. The protein expression levels of TRPC5/7 were only affected by 4 ng/mL and 8 ng/mL TGF-β1, respectively. TGF-β1 increased TRPC1/3/6 mRNA levels in podocytes, however had no effects on TRPC4/5/7 mRNA. TGF-β1 significantly increased [Ca2+]i in podocytes. Knockdown of TRPC1/4/5/7 in podocytes had no significant effect on the [Ca2+]i induced by TGF-β1, but TRPC3/6 knockdown significantly decreased the [Ca2+]i. There was no significant difference in the [Ca2+]i between the TRPC6 siRNA-treated group and SKF96365-treated group, but the [Ca2+]i of the TRPC3 siRNA-treated group was significantly higher than that of SKF96365-treated group. These results demonstrate that TGF-β1 increases the expression of the TRPC1/3/6 in podocytes. TGF-β1 increases [Ca2+]i in podocytes, which is dependent on the TRPC3/6 expression. Our results also suggest that the effect of TRPC6 on [Ca2+]i in podocytes may be greater than that of TRPC3.

Interventional effect of asiaticosdide on rats exposed to silica dust / 中华劳动卫生职业病杂志

Objective: To investigate the effect of asiaticoside for fibrosis in lung tissues of rats exposed to silica and to explore its possible mechanism. Methods: 144 SD male rats were randomly divided into control group, model group, positive drug control group, asiaticoside high-dose group, medium-dose group and low-dose group, each group included 24 rats. Rats in the control group were perfused with 1.0 ml of normal saline, and the other groups were given 1.0 ml 50 mg/ml SiO(2) suspension. Gavage of herbal was given from the next day after model establishment, once a day. Rats in the positive drug control group were administration with 30 mg/kg tetrandrine and rats in the low-dose group, medium-dose group and high-dose group were given 20 mg/kg, 40 mg/kg and 60 mg/kg asiaticoside for fibrosis respectively. Rats in the control group and the model group were given 0.9% normal saline. The rats were sacrificed in on the 14th, 28th and 56th day after intragastric administration and collect the lung tissues to detect the content of hydroxyproline, TGF-β(1) and IL-18, observe the pathological changes of the lung tissues by HE and Masson staining and determine the expressions of Col-I, a-SMA, TGF-β in lung tissues by Western Blot. Results: On the 14th day, 28th day and 56th day after model establishment, the lung tissues of rats in the model group showed obvious inflammatory response and accumulation of collagen fibers, and the degree of inflammation and fibrosis increased with time. The intervention of asiaticoside could effectively inhibit the pathological changes of lung tissues. The contents of hydroxyproline, IL-18 and TGF-β1 in lung tissues of model group were higher than those in the control group (P<0.05) , while the level of hydroxyproline, IL-18 and TGF-β1 in asiaticoside groups were significantly decreased, and the difference was statistically signicant (P<0.05) . Compared with the control group, the expression levels of Col-I, TGF-β1and α-SMA in lung tissue of model group were increased (P<0.05) , while the expression level of Col-I, TGF-β1 and α-SMA were decreased after the intervention of asiaticoside, and the difference was statistically signicant (P<0.05) . Conclusion: Asiaticoside can inhibit the increase of Col-I, TGF-β1 and α-SMA content in the SiO(2)-induced lung tissues of rats, reduce the release of TGF-β1 and IL-18 inflammatory factors in lung tissue, and then inhibit the synthesis and deposition of extracellular matrix in rat lung tissue, and improve silicosis fibrosis.

Dihydromyricetin mediates epithelial mesenchymal transformation and regulates the proliferation and apoptosis of esophageal squamous cell carcinoma cells / 中华肿瘤杂志

Objective: To study the effects of dihydromyricetin (DMY) on the proliferation, apoptosis and epithelial mesenchymal transition (EMT) of esophageal squamous cell carcinoma (ESCC) cell KYSE150 and KYSE410. Methods: KYSE150 and KYSE410 cells were treated with different concentrations of DMY (0, 25, 50, 100, 150, 200 μmol/L) for 24 hours. The median inhibition concentration (IC50) values of KYSE150 and KYSE410 were detected by cell counting kit-8 (CCK-8) method. Then 0.5‰ dimethyl sulfoxide (DMSO) was used as control group, dihydromyricetin (DMY), dihydromyricetin and transforming growth factor-β1 (DMY+ TGF-β1), transforming growth factor-β1 (TGF-β1) were used as experimental group. Cell proliferation and apoptosis rates were measured by clonal formation and flow cytometry. Transwell invasion and wound healing assay were used to detect cell invasion and migration. The protein expression levels of Caspase-3, Caspase-9, Bcl-2, Bax, Smad2/3, phosphorylation-Smad2/3 (p-Smad2/3) and Vimentin were detected by western blot. Results: The IC50 values of DMY on KYSE410 and KYSE150 cells were 100.51 and 101.27 μmol/L. The clone formation numbers of KYSE150 and KYSE410 in DMY group [(0.53±0.03) and (0.31±0.03)] were lower than those in DMSO group [(1.00±0.10) and (1.00±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in DMY group [(1.84±0.22)% and (2.80±0.07)%] were higher than those in DMSO group [(1.00±0.18)% and (1.00±0.07)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in DMY group [(0.42±0.03) and (0.29±0.05)] were lower than those in DMSO group [(1.00±0.08) and (1.00±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in DMY group [(0.65±0.14)% and (0.40±0.17)%] were lower than those in DMSO group [(1.00±0.10)% and (1.00±0.08)%, P<0.05]. The clone formation numbers of KYSE150 and KYSE410 in TGF-β1 group [(1.01±0.08) and (0.99±0.25)] were higher than those in DMY+ TGF-β1 group [(0.73±0.10) and (0.58±0.05), P<0.05]. The apoptosis rates of KYSE150 and KYSE410 cells in TGF-β1 group [(0.81±0.14)% and (1.18±0.10)%] were lower than those in DMY+ TGF-β1 group [(1.38±0.22)% and (1.85±0.04)%, P<0.05]. The invasion numbers of KYSE150 and KYSE410 cells in TGF-β1 group [(1.19±0.11) and (1.39±0.11)] were higher than those in DMY+ TGF-β1 group [(0.93±0.09) and (0.93±0.05), P<0.05]. The migration rates of KYSE150 and KYSE410 cells in TGF-β1 group [(1.87±0.19)% and (1.32±0.04)%] were higher than those in DMY+ TGF-β1 group [(0.86±0.16)% and (0.77±0.12)%, P<0.05]. The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY group were higher than those in DMSO group, while the protein expression level of Bcl-2 was lower than that in DMSO group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in DMY group were lower than those in DMSO group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in TGF-β1 group were lower than those in DMY+ TGF-β1 group, and the protein expression level of Bcl-2 was higher than that in DMY+ TGF-β1 group (P<0.05). The protein expression levels of Bax, Caspase-3 and Caspase-9 in KYSE150 and KYSE410 cells in DMY+ TGF-β1 group were lower than those in DMY group, and the protein expression level of Bcl-2 was higher than that in DMY group (P<0.05). The protein expression levels of p-Smad2/3, Smad2/3 and Vimentin in KYSE150 and KYSE410 cells in TGF-β1 group were higher than those in DMY+ TGF-β1 group (P<0.05). Conclusion: DMY can inhibit the proliferation and EMT of ESCC mediated by TGF-β1 and promote cell apoptosis.

Inhibition of Sonic Hedgehog signaling inhibits fibrous scar formation and adversely affects functional outcome after ischemic brain injury in rats / 南方医科大学学报

OBJECTIVE@#To investigate the effects of inhibiting Sonic Hedgehog (Shh) signaling on fibrous scar formation and functional outcome after ischemic brain injury.@*METHODS@#Adult SD rats were randomized into sham-operated group, middle cerebral artery occlusion (MCAO) and reperfusion (I/R) group, I/R with intraventricular empty adenoviral vector (rAd-NC) injection group, and I/R with adenovirus-mediated Shh knockdown (rAd-ShShh) group. After the treatments, the neurological deficits of the rats were assessed, and the protein and mRNA expressions of fibronectin (Fn), α-SMA, and Shh in the ischemic hemisphere were detected with immunofluorescence assay and qPCR; TUNEL staining was used for detecting neural cell apoptosis. In the cell experiment, primary meningeal fibroblasts isolated from neonatal SD rats were pretreated for 24 h with TGF-β1 or TGF-β1 plus cyclopamine (CYC) before oxygen-glucose deprivation for 150 min followed by reoxygenation for 72 h (OGD/R). CCK-8 assay and scratch test were performed to examine the changes in cell proliferation and migration, and immunofluorescence assay, qPCR and Western blotting were used for detecting cell transformation and the expressions of Shh, α-SMA, and Fn.@*RESULTS@#Cerebral I/R injury significantly increased the protein and mRNA expressions of Shh, α-SMA, and Fn in the ischemic hemisphere of the rats, but their expression levels were significantly lowered by intraventricular injection of rAd-Shshh (P < 0.05), which obviously increased cell apoptosis in the ischemic hemisphere (P < 0.05) and improved modified mNSS and modified Bederson scores of the rats (P < 0.05). In the cell experiment, pretreatment with TGF-β1 and TGF-β1+CYC both increased the viability of the primary meningeal fibroblasts after OGD/R. TGF-β1 significantly enhanced the migration ability and induced obvious transformation of the exposed cells (P < 0.05), but these effects were significantly attenuated by co-treatment with CYC (P < 0.05). The expressions of Shh, α-SMA and Fn in the TGF-β1 group were all significantly higher in TGF-β1-treated cells (P < 0.05) and were obviously lowered by co-treatment with CYC (P < 0.05).@*CONCLUSION@#Inhibition of Shh signaling may inhibit fibrous scar formation and functional recovery in rats after ischemic brain injury.