苹果乙烯响应因子基因MdERF11对脱落酸的敏感性分析

以‘嘎拉’苹果（Malus × domestica Borkh.）为试材,克隆了乙烯响应因子基因MdERF11（序列号 MDP0000756341）。测序发现,该基因包含全长为483 bp的完整开放阅读框,编码161个氨基酸。系统进化树分析表明,这一乙烯响应因子与拟南芥AtERF11蛋白同源序列相似性最高。利用PlantCare数据库进行基因启动子顺式作用元件预测,MdERF11启动子序列中含有与脱落酸（ABA）、乙烯及干旱信号相关的顺式作用元件。荧光定量PCR分析表明,MdERF11在苹果的各组织中均有表达,在叶柄和果实中表达量相对较高;并且MdERF11的表达明显受到ABA的诱导。在外源ABA的处理下,MdERF11过量表达的苹果愈伤组织的生长势明显比野生型强,表明MdERF11降低了苹果愈伤组织对ABA的敏感性。     

Activation of macrophage peroxisome proliferator-activated receptor α inhibits macrophage inflammation-induced cardiac fibroblast activation and migration

AIM: To investigate the effect of macrophage peroxisome proliferator-activated receptor α (PPARα) activation on macrophage inflammation-induced activation and migration of cardiac fibroblasts. METHODS: Mouse bone marrow-derived macrophages were treated with vehicle, PPARα agonist WY14643 (10 μmol/L), angiotensin Ⅱ (Ang II; 1 μmol/L) or Ang II+WY14643 for 24 h, and the supernatants were collected as conditioned medium (CM) to stimulate cardiac fibroblasts for additional 24 h. The mRNA levels of PPARα, interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in the macrophages as well as fibrotic markers collagen type Ⅰ alpha 2 chain (Col1a2), collagen alpha 1 chain (Col3a1) and actin alpha 2 (Acta2) in the cardiac fibroblasts were detected by RT-qPCR. The protein levels of IL-6 and IL-1β in the macrophages as well as collagen I, collagen III and α-smooth muscle actin (α-SMA; encoded by Acta2 gene) in the cardiac fibroblasts were determined by Western blot. Wound-healing assay was applied to eva-luate the migration ability of cardiac fibroblasts. RESULTS: Ang II significantly increased the mRNA levels of pro-inflammatory factors, such as IL-6, IL-1α and TNF-α, but decreased the mRNA level of PPARα in the macrophages. Administration of PPARα agonist WY14643 dramatically decreased Ang II-induced mRNA levels of IL-6, IL-1β and TNF-α in the macrophages, and significantly decreased Ang II-induced protein expression of IL-6 and pro-IL-1β in the macrophages. The CM from Ang II-treated macrophages significantly up-regulated the mRNA levels of Col1a2, Col3a1 and Acta2 in the cardiac fibroblasts, which were inhibited by the CM from WY14643-treated macrophages. The same results were observed in the protein levels of collagen I, collagen III and α-SMA in the cardiac fibroblasts. Moreover, the CM from Ang II-treated macrophages significantly promoted cardiac fibroblast migration, whereas the CM from WY14643-treated macrophages markedly inhibited macrophage inflammation-induced cardiac fibroblast migration. CONCLUSION: WY14643-activated PPARα inhibits activation and migration of cardiac fibroblasts by attenuating Ang II-induced macrophage inflammatory response.     

Effect of angiotensin-(1-7) on angiotensin II-induced rat renal interstitial fibroblast activation

AIM: To explore the influence of angiotensin-(1-7) on angiotension II (Ang II)-induced activation and extracellular matrix secretion in rat renal interstitial fibroblasts (NRK-49F cells). METHODS: The NRK-49F cells were maintained and sub-cultured, then the cells were divided into control group, Ang II group, Ang-(1-7) group and Ang II+Ang-(1-7) group. The expression of α-smooth muscle actin(α-SMA),transforming growth factor β₁ (TGF-β₁) and insulin-like growth factor I(IGF-I) was detected by the method of immunocytochemistry when the cells were cultured for 72 h. The content of TGF-β₁, IGF-I and collagen type I(Col I) in the cultured supernatants were measured by ELISA. RESULTS: In control group and Ang-(1-7) group, only basic expression of α-SMA and almost no expression of TGF-β₁, IGF-I and Col I were observed. Compared with control group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was increased in Ang II group. Compared with Ang II group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was significantly decreased in Ang II+Ang-(1-7) group.CONCLUSION: Ang-(1-7) inhibits the activation of renal interstitial fibroblasts and decreases the Ang II induced secretion of Col I by suppressing TGF-β₁ and IGF-I expression.     

Aerobic exercise protects cardiac function of T2DM mice by activation of PI3K (p110α)/Akt signaling pathway

AIM: To study the protective effect of aerobic exercise on cardiac dysfunction in mice and its mechanism, and to provide theoretical and practical basis for the exercise therapy of diabetic cardiac dysfunction.METHODS: The mice were divided into normal control non-exercise (NNC) group, normal control exercise (ENC) group, diabetic non-exercise (NDM) group and diabetic exercise (EDM) group. At the end of the experiment, the cardiac function was evaluated by echocardiography. The pathological changes of the myocardial tissues and the development of fibrosis were observed. The mRNA expression of ANP, and the protein levels of PI3K (p110α) and Akt were determined. RESULTS: The decrease in cardiac function of diabetic mice was observed, and the cardiac function recovered after exercise intervention (P<0.05). Under light microscope with HE and Masson staining, the myocardial structure in NDM group was in extreme disorder, cell arrangement was not neat, and the degree of fibrosis increased, but the myocardial damage was improved in ENC group. Compared with NNC group, the mRNA expression of ANP in the myocardium of diabetic mice was up-regulated (P<0.05). The protein levels of PI3K (p110α) and Akt were decreased (P<0.05), and the cascade was inactivated. Compared with NDM group, the mRNA expression of ANP was down-regulated and the protein levels of PI3K (p110α) and Akt were up-regulated in EDM group (P<0.05). CONCLUSION: Diabetes results in myocardial damage in mice, and reduces cardiac function. Exercise intervention alleviates the heart dysfunction induced by high glucose via activating PI3K(p110α)/Akt signaling pathway to protect the structure and function of the myocardium.     

Gefitinib inhibits epithelial-mesenchymal transition by up-regulating Foxo3a expression in mice with bleomycin-induced lung fibrosis

AIM：To identify the effect of gefitinib on the expression of forkhead box protein O3a (Foxo3a), α-smooth muscle actin (α-SMA) and related signal pathway molecules in the mice with bleomycin-induced lung fibrosis and to investigate the inhibition mechanism of gefitinib on lung epithelial-mesenchymal transition. METHODS：Thirty Kunming female mice were randomly divided into 3 groups:control group (received normal saline intratracheally), bleomycin group (received bleomycin intratracheally, 3 mg/kg), and bleomycin plus gefitinib group (received bleomycin intratracheally and gefitinib orally, 20 mg/kg). All the mice were sacrificed 14 d after the treatments. Pulmonary histological changes were evaluated by hematoxylin-eosin staining and Masson trichrome staining. The mRNA levels of Foxo3a and α-SMA in the lung tissues were detected by RT-PCR. Nuclear Foxo3a, α-SMA, and phosphorylation of EGFR, Akt and Foxo3a in the lung tissues were determined by Western blotting. RESULTS：Gefitinib inhibited bleomycin-induced lung fibrosis and significantly decreased the scores of lung inflammation and fibrosis. Foxo3a mRNA expression and total Foxo3a protein expression were increased, while the phosphorylated Foxo3a was decreased. Nuclear Foxo3a was increased significantly. Meanwhile, phosphorylated EGFR and Akt were decreased. The level of α-SMA was observably increased. CONCLUSION:Gefitinib restores Foxo3a activity and reduces α-SMA expression by modulating EGFR/Akt activity, thus inhibiting bleomycin-induced lung fibrosis.     

Chronic alcohol intake-induced epithelial-mesenchymal transition contri-butes to hepatic fibrogenesis in mice

AIM：To evaluate the effect of chronic alcohol intake on the histopathological changes of the liver and to determine the contribution of epithelial-mesenchymal transition (EMT) to hepatic fibrogenesis. METHODS：Thirty male C57BL/6 mice were randomly divided into 3 groups as following: the mice in control group was given (ig) water; the mice in low-dose alcohol group (2.0 g·kg -1·d -1) and high-dose alcohol group (4.0 g·kg -1·d -1) were given (ig) alcohol for 5 months. Alcohol-induced histopathological changes of the liver or development of hepatic fibrosis were evaluated using the histological methods with HE and Masson trichrome staining. The apoptosis of the liver was detected by TUNEL fluorometric staining (counterstained with DAPI). The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was measured by an automated biochemical analyzer. The expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA) and E-cadherin in the hepatic tissues was detected by immunofluorescence examination. The protein levels of E-cadherin, α-SMA, FSP-1, transforming growth factor β 1 (TGF-β 1) and hypoxia-inducible factor 1α (HIF-1α) were analyzed by Western blotting. RESULTS：Compared with control, the activity of serum ALT and AST, and apoptotic index of liver tissues were increased in the mice treated with alcohol for 5 months. The histopathological changes of the livers in the mice of low-dose alcohol group included steatosis and mild liver fibrosis, while severe liver fibrosis was observed in the high-dose alcohol-treated mice. Chronic alcohol consumption induced the increase in malondialdehyde (MDA) level, and the decreases in the activity of superoxide dismutase (SOD) and catalase (CAT) in the livers. It also reduced E-cadherin expression and increased α-SMA expression. FSP-1 immunostaining and albumn immunostaining positive cells were co-localized in the hepatocytes of low-dose alcohol group, but only FSP-1 positive hepatocytes were observed in high-dose alcohol group. Chronic alcohol consumption decreased E-cadherin expression and increased α-SMA, FSP-1, TGF-β 1 and HIF-1α expression in a dose-dependent manner, but the HIF-1α expression was not altered between the 2 alcohol-treated groups. CONCLUSION：Chronic alcohol intake induces the progression of hepatic fibrosis. Some fibroblasts derive from hepatocytes in liver fibrosis via EMT. The underlying mechanism is associated with the changes of the redox state, and increased TGF-β 1 generation and HIF-1α expression.     

Effect of all-trans-retinoic acid on the proliferation and expression of α-SMA in human embryonic lung fibroblasts

AIM: To investigate the effects of all-trans-retinoic acid (ATRA) on the proliferation and differentiation of transforming growth factor β₁(TGF-β₁)-stimulated human embryonic lung fibroblasts (HFL-I).METHODS: The HFL-I cells were cultured in vitro and were pretreated with ATRA for 3 days at the concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L. The proliferation of HFL-1 cells was detected by MTT method. The mRNA expression of α-smooth muscle actin(α-SMA) in HFL-I cells stimulated with TGF-β₁ for 0 h, 6 h, 12 h, 24 h, 48 h and 72 h was detected by RT-PCR and the protein expression of α-SMA at the time points of 1,3 and 5 days was detected by Western blotting. The mRNA expression of α-SMA in HFL-I cells pretreated with different concentrations of ATRA for 24 h was detected the by RT-PCR and the protein expression at time point of 3rd day was detected by Western blotting. RESULTS: Different concentration of ATRA inhibited the proliferation of HFL-I in a dose-dependent manner (P<0.05). Both mRNA and protein expression of α-SMA in HFL-I cells pretreated with TGF-β₁ was up-regulated (P<0.05). ATRA down-regulated the mRNA and protein expression of α-SMA induced by TGF-β₁ in a dose-dependent manner (P<0.05). CONCLUSION: ATRA inhibits the proliferation and TGF-β₁-stimulated differentiation in HFL-I cells by down-regulating the mRNA and protein expression of α-SMA.     

Study on relationship between expression of PKCα in glomeruli and development of nephropathy in diabetic rats

AIM:To observe the dynamic changes of expression of PKCα, TGF-β₁ and α-SMA in glomeruli of diabetic rats induced by the alloxon and to invesitigate their roles in the diabetic nephropathy(DN).METHODS:Rats were randomly divided into four groups: normal control group (group A), diabetic group of one week (group B), diabetic group of one month (group C), diabetic group of two months (group D). Immunohistochemistry and Western blotting were used to detect the expression of PKCα, TGF-β₁ and α-SMA in renal tissue of all groups. Blood glucose, triglycerides, cholesterol, creatinine and urine protein were analysed by chemical methods. The morphological changes of renal tissue were checked through microscopy.RESULTS:The expression of PKCα and TGF-β₁ in renal tissue of diabetic groups were increased comparing with those of nomal control group(P<0.05). The mesangial cells expressed α-SMA in two months group. Chronologically the expression of PKCα, TGF-β₁ and α-SMA were positively correlative with each other and the impairment of kidney was also observed.CONCLUSIONS: During the DN process the expression of PKCα increased. PKCα raised GFR and the permeability of glomerular filtration membrane which enhanced urinary albumin excretion. PKCα also increased expression of TGF-β and therefore to induce the expression of α-SMA. The appearance of α-SMA was a marker of the phenotypic transform of renal cells.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

Effects of epithelial cells treated with poly (I:C) on activation of fibroblasts and roles of EMMPRIN

AIM: To investigate the effects of epithelial cells treated with polyinosinic-polycytidylic acid [poly(I:C)] on the proliferation, transdifferentiation and signaling mechanisms of airway fibroblasts.METHODS:Human alveolar epithelial cells were treated with poly(I:C). The cell culture supernatants were used to stimulate the airway fibroblasts or the fibroblasts growing in collagen gels. The proliferation of the fibroblasts, the expression of a-smooth muscle actin (α-SMA) in the fibroblasts and the contractility of the collagen gels containing fibroblasts, as well as the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) were observed. The proliferation of the fibroblasts and the expression levels of α-SMA, MMP-2 and MMP-9 in the fibroblasts stimulated with EMMPRIN were detected. The inhibitors specific for p38 MAPK or ERK1/2 were used to explore the effects on α-SMA expression and EMMPRIN secretion. RESULTS:The culture supernatants of the epithelial cells treated with poly(I:C) induced the proliferation, α-SMA expression and gel contraction as well as EMMPRIN secretion in the fibroblasts. EMMPRIN dose-dependently enhanced fibroblast proliferation, α-SMA expression and activity of MMP-2 and MMP-9. The supernatants of epithelial cells treated with poly(I:C) activated p38 MAPK and ERK1/2 signaling in the fibroblasts, as the specific inhibitors of MAPK or ERK1/2 signaling attenuated the expression of α-SMA and EMMPRIN in the fibroblasts. CONCLUSION: Poly(I:C) induces fibroblast proliferation, α-SMA expression and gel contraction by affecting the process mediated by p38 MAPK and ERK1/2 signaling pathways in epithelial cells. EMMPRIN may be the important media involved in this event.     

PAX6 inhibits angiotensin II-induced cardiac fibroblast transdifferentiation

AIM To investigate the effects of paired box 6 （PAX6） on angiotensin II （Ang II）-induced transdifferentiation of cardiac fibroblasts （CFs） and its underlying mechanisms. METHODS Primary CFs were isolated from the hearts of adult mice, and Ang II was used to induce the transdifferentiation of CFs. The adenovirus vector carrying PAX6 was constructed and transfected into the CFs. The cells were divided into Ad-GFP+Ctrl group （transfected with control adenovirus vector）, Ad-GFP+Ang II group （transfected with control adenovirus vector and treated with Ang II）, Ad-PAX6+Ctrl group （transfected with adenovirus vector carrying PAX6） and Ad-PAX6+Ang II group （transfected with adenovirus vector carrying PAX6 and treated with Ang II）. The fluorescence expressed by transfected CFs was observed under an inverted fluorescence microscope. The protein levels of PAX6, α-smooth muscle actin （α-SMA）, collagen type I （Col I）, fibronectin （FN） and transforming growth factor β1 （TGFβ1） were detected by Western blot. The expression and distribution of α-SMA, Col I and FN were measured by immunofluorescence staining. The mRNA levels of PAX6 and TGFβ1 were determined by qPCR. RESULTS The fluorescence observed by inverted fluorescence microscopy confirmed the successful transfection of adenovirus vector into the CFs. qPCR and Western blot showed successful PAX6 overexpression in the CFs （P<0.01）. Ang II increased the myofibroblast marker α-SMA in the CFs, while overexpression of PAX6 significantly inhibited the expression of α-SMA induced by Ang II （P<0.01）. In addition, PAX6 overexpression significantly inhibited Ang II-induced synthesis of extracellular matrix （ECM） proteins Col I and FN （P<0.05）. Furthermore, Ang II treatment upregulated TGFβ1 mRNA and protein levels, while overexpression of PAX6 significantly inhibited TGFβ1 mRNA and protein expression induced by Ang II （P<0.05）. CONCLUSION PAX6 inhibits Ang II-induced CF transdifferentiation and ECM protein synthesis via inhibiting TGFβ1 expression.     

Activation of α7nAChR promotes wound healing in diabetic mice by suppressing TNF-α expression

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR)-specific agonist PNU-282987 in promoting wound healing in diabetic mice by suppressing the expression of tumor necrosis factor α （TNF-α）.METHODS: A model of incised wound was established in diabetic mice or normoglycaemic mice (control). Skin samples were taken on 1 d, 3 d, 5 d, 10 d, 14 d and 21 d post-injury (5 mice in each posttraumatic interval). The numbers of macrophages and fibroblasts, the expression of TNF-α and the deposition of collagen were detected by the methods of immunohistochemistry, Western blotting and Masson staining, respectively. After incised wound was performed in the diabetic mice, PNU-282987 was applied by intraperitoneal injection at suitable posttraumatic interval. The above indexes were investigated again.RESULTS: Compared with control group, the diabetic mice presented delayed wound healing. In diabe-tic mice, the infiltration of macrophages and the expression of TNF-α were significantly reduced in the early phase during wound healing, while they were significantly increased from 5 d post-injury. Besides, from 5 d to 21 d post-injury, the wounds in diabetic mice showed decreased number of fibroblasts and deposition of collagen. From 5 d post-injury, PNU-282987 was applied to diabetic mice, which significantly down-regulated the expression of TNF-α, and increased the number of fibroblasts and the content of collagen in the wounds, eventually promoted wound healing.CONCLUSION: Inflammatory reactions delay wound healing in diabetic mice. Activation of α7nAChR promotes wound healing in diabetic mice by suppressing the expression of TNF-α.     

Role of nucleolin in Ang II-induced phenotypic transformation of vascular smooth muscle cells

AIM: To investigate the effect of nucleolin on angiotensin II (Ang II)-induced phenotypic transformation of vascular smooth muscle cells (VSMCs). METHODS: Ang II was used to induce the phenotypic transformation of VSMCs. The spatial and temporal expression patterns of nucleolin, and the effects of Ang II on the expression of VSMC phenotypic transformation markers α-smooth muscle actin (α-SMA), calponin, smooth muscle protein 22 α (SM22α) and osteopontin (OPN) were investigated. The techniques of gene over-expression and RNA interference were used to assess the effect of nucleolin on the expression of Ang II-mediated VSMC phenotypic transformation markers. RESULTS: The expression of α-SMA, SM22α and calponin at the mRNA and protein levels was gradually decreased by Ang II stimulation, while the expression of OPN at mRNA and protein levels was gradually increased. The expression of nucleolin was gradually up-regulated in the VSMCs treated with Ang II at different concentrations for various duration (P<0.05). Ang II induced nucleolin translocation from the nucleus to cytoplasm. Over-expression of nucleolin promoted the VSMC phenotypic transformation induced by Ang II. Down-regulation of nucleolin suppressed the promotion of phenotypic transformation. CONCLUSION: Nucleolin promotes Ang II-induced phenotypic transformation of VSMCs, and its mechanism may be related to its function of cytoplasmic translocation.     

Effect of c-SKI on coronary endothelial cell proliferation and endothelial-mesenchymal transition

AIM: To investigate the effect of cellular Sloan-Kettering Institute (c-SKI) on the proliferation and endothelial-mesenchymal transition of human coronary artery endothelial cells (HCAECs). METHODS: HCAECs were treated with transforming growth factor-β1 (TGF-β1) at varying concentrations for different time points. Western blot was used to test the expression of c-SKI and mesenchymal markers such as α-smooth muscle actin (α-SMA) and vimentin. Meanwhile, the endothelial marker E-cadherin was also detected. HCAECs were transfected with c-ski gene mediated by lentivirus (LV), the efficiency of LV-SKI transfection was detected by RT-qPCR. The HCAECs were divided into 4 groups:control group, TGF-β1 (5 μg/L) group, LV-SKI+ TGF-β1 group, LV-NC+ TGF-β1 group. The cell viability and colony formation were measured by MTT assay and colony formation assay. The protein levels of vimentin, α-SMA, E-cadherin, Smad2, Smad3, p-Smad2 and p-Smad3 were determined by Western blot. RESULTS: The expression of c-SKI was down-regulated in the HCAECs treated with TGF-β1 (P<0.01). Over-expression of c-SKI inhibited the proliferation of HCAECs (P<0.01). Compared with LV-NC group, over-expression of c-SKI down-regulated the expression of α-SMA and vimentin (P<0.01), up-regulated the expression of E-cadherin (P<0.01), and inhibited the protein phosphorylation of Smad2 and Smad3 (P<0.01), reversed the endothelial-mesenchymal transition induced by TGF-β1. CONCLUSION: The expression of c-SKI in the HCAECs is down-regulated in the process of endothelial-mesenchymal transition. Over-expression of c-SKI inhibits proliferation and endothelial-mesenchymal transition of HCAECs, the mechanism may be related to regulation of the TGF-β1/Smad signaling pathway.     

Changes of histone modification levels in rats with liver fibrosis

AIM: To observe the changes of histone modifications in the liver of rats with hepatic fibrosis and its possible role in the development of hepatic fibrosis. METHODS: Male Wistar rats (n=20) were randomly divided into liver fibrosis group and normal control group. The liver fibrosis model was established by hypodermic injection of CCl₄, and the rats in normal control group were injected with vegetable oils. At the end of the 8th week, all rats were killed. Liver function indexes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and liver fibrosis indexes including haluronic acid (HA), laminin (LN), collagen type IV (Col Ⅳ) and procollagen type Ⅲ (PCⅢ) were determined by biochemical and RIA methods. The liver index was analyzed, and the liver fibrosis degree and the morphological change of the liver were detected by HE and Masson staining. The levels of α-smooth muscle actin (α-SMA), collagen type Ⅰ (ColⅠ), H3K4me2, H3K9me2, acH3K9 and acH4K12 were detected by Western blot. RESULTS: After hypodermic injection of CCl₄ for 8 weeks, the liver index, ALT, AST, HA, LN, Col Ⅳ and PCⅢ of the rats in liver fibrosis group were higher than those in control group (P<0.05). Compared with control group, the level of acH4K12 was decreased (P<0.05), while acH3K9, H3K9me2, α-SMA and ColⅠ were increased (P<0.05), but H3K4me2 had no significant change.CONCLUSION: The levels of acH4K12, acH3K9 and H3K9me2 may play essential roles in the pathogenesis of liver fibrosis, and these histone modifications may regulate gene expression associated with extracellular matrix metabolism.     

Effect of resveratrol on autophagy and renal interstitial fibrosis in kidney of diabetic mice

AIMTo observe the effect of resveratrol (Res) on renal autophagy level and renal interstitial fibrosis in the mice with diabetes mellitus (DM), and to discuss the possible mechanism. METHODSThe wild-type C57BL/6 mice were randomly divided into 3 groups, including normal control (NC) group, DM group and Res group (8 in each group). The diabetic mouse model was established by injection of streptozotocin. After 8 weeks of successful replication of the diabetic model, Res was given to the mice in Res group by continuous gavage for 12 weeks, and then the mice in each group were sacrificed to detect the relevant biochemical parameters. The pathological changes of the kidney tissues were observed by HE staining and Masson staining. The levels of the proteins related to autophagy, epithelial-mesenchymal transition (EMT) and fibrosis were determined by Western blot. The mRNA expression of collagen type IV (Col IV), α-smooth muscle actin (α-SMA) and E-cadherin were detected by real-time PCR. RESULTSCompared with NC group, fasting blood glucose (FBG), kidney index (KI), serum creatinine, 24-hour urinary albumin excretion rate and 24-hour urine total protein were remarkably increased in DM group (P<0.05). The results of HE and Masson staining indicated that renal tissue presented fibrosis in DM group. The protein levels of E-cadherin, beclin-1, microtubule-associated protein 1 light chain 3-II (LC3-II) were reduced in DM group, while the levels of α-SMA, Col IV and Snail1 were increased (P<0.05). After intervention with Res for 12 weeks, all the relevant biochemical parameters and KI were reduced (P<0.05) except FBG (P>0.05), and renal fibrosis lesions were obviously alleviated. Compared with DM group, the protein levels of E-cadherin, beclin-1 and LC3-II were increased in Res group, but the protein expression levels of α-SMA, Col IV, Snail1 were reduced (P<0.05). Compared with DM group, the mRNA level of E-cadherin was increased in Res group , but the mRNA levels of Col IV and α-SMA were reduced (P<0.05). CONCLUSION Resveratrol significantly inhibits EMT and reduces renal interstitial fibrosis in diabetic mice, and its mechanism may be related to the promotion of renal autophagy.     

Increased fucosyltransferase 8 expression in rat kidney with unilateral ureteral obstruction

AIM: To investigate the effect of fucosyltransferase 8 (FUT8) on rat renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). METHODS: Ninety male Wistar rats were randomly divided into normal control group (control), sham operation group (sham) and UUO group, and sacrificed on day 1, 3, 7, 14 and 21 after operation. Serum creatinine and urea nitrogen were detected to assess renal function. PAS and Masson staining were used to observe histological changes of the rat kidneys. The time-correlated expression of FUT8 in the kidney was monitored by RT-PCR and Western blotting. The protein expression levels of FUT8 and activin receptor-like kinase 5(ALK5) were determined by immunofluorescence double-staining. Immunohistochemical staining was also used to assess the protein expression of fibronectin (FN), type I collagen (Col I) and α-smooth muscle actin (α-SMA). RESULTS: Compared with control group, serum creatinine and urea nitrogen in UUO group elevated on day 3 after operation (P<0.05) and reached the peak value on day 21 after operation (P<0.01). Renal tubule atrophy and renal interstitial fibrosis were observed in UUO group 7 and 14 days after operation. The mRNA and protein expression levels of FUT8 increased markedly in UUO group on day 3 (P<0.05) and reached its peak value on day 14 and 21 after operation (P<0.01). The results of immunofluorescence and immunohistochemistry showed that FUT8 and ALK5 were coexpressed in renal tubulointestitium. FN, Col I and α-SMA were significantly elevated in UUO group (P<0.05), and were positively correlated with the expression of FUT8 (P<0.05).CONCLUSION: The expression of FUT8 influences the progress of renal interstitial fibrosis, tubule atrophy and inflammatory cell infiltration in the kidney.     

VPA alleviates bleomycin-induced pulmonary fibrosis in rats

AIM: To investigate the effect of sodium valproate (VPA) on bleomycin-induced pulmonary fibrosis (PF) and its mechanism. METHODS: SD rats (n=42) were randomly divided into model group and treatment group. Bleomycin at dose of 5 mg/kg was intratracheally injected to establish a rat PF model. The rats in treatment group were given normal saline (NS, 0.5 mL/d), VPA (300 mg·kg^-1·d^-1) or dexamethasone (DEX, 0.6 mg·kg^-1·d^-1) via intraperitoneal injection from the 14th day to the 28th day after modeling. The rats in model group were sacrificed on 0 d, 14 day and 28 d after modeling . The rats in treatment group were killed at 14th day after treatment. The effects of VPA on PF were evaluated by HE and Masson staining. The hydroxyproline (HYP) content in the rat lung tissues was detected, and the expression of α-smooth muscle actin (α-SMA) and E-cadherin was determined by Western blotting. RESULTS: HE staining showed that the alveolar structure and interstitial morphology in VPA group were better than those in NS group and DEX group. The level of collagen in VPA group was significantly lower than that in DEX group and NS group by determining the HYP content and Masson staining. VPA reduced the expression of α-SMA, a mesenchymal marker protein of PF, while increased the expression of epithelial marker protein E-cadherin. CONCLUSION: VPA reduces collagen content and distribution, and up-regulates the expression of the epithelial marker protein E-cadherin, while down-regulates mesenchymal marker protein α-SMA, thereby preventing the rat lung tissues from bleomycin-induced PF.     

Imatinib attenuates DOCA/salt-induced rat myocardial fibrosis via PDGFs/PDGFRs signaling pathway

AIM：To investigate the role of imatinib (IMA) in reducing myocardial fibrosis and its relationship with platelet-derived growth factors (PDGFs)/PDGF receptors (PDGFRs) signaling pathway in deoxycorticosterone acetate (DOCA)/salt-induced hypertension in rats. METHODS:Sixty male uninephrectomized SD rats were treated with 1% NaCl and 0.2% KCl in the drinking water for 4 weeks and randomly divided into vehicle control (CON) group, DOCA treatment (DOCA) group and DOCA plus IMA treatment (DOCA+IMA) group. Systolic blood pressure (SBP) was mea-sured weekly using the tail-cuff method. The extent of myocardial interstitial fibrosis and the ratio of perivascular collagen area/vascular area (PVCA/VA) were analyzed by picro-Sirius red staining. The mRNA expression of fibroblast-specific protein 1 (FSP-1), α-smooth muscle actin (α-SMA), procollagenⅠ, procollagenⅢ, PDGFRα and PDGFRβ in the myocardium was measured by real-time PCR. The protein levels of PDGFRα, PDGFRβ, p-PDGFRβ, vimentin and α-SMA were analyzed by immunohistochemical staining. The cell types of PDGFRα and PDGFRβ localizations were examined by immunofluorescence method. RESULTS：(1) SBP in DOCA and DOCA+IMA groups was higher than that in CON group on day 14 and day 28, and no difference of SBP between DOCA group and DOCA+IMA group was found. The extent of myocardial interstitial fibrosis and the ratio of PVCA/VA in DCOA group were higher than those in CON group, and those in DOCA+IMA group were significantly lower than those in DOCA group on day 28. (2) Compared with CON group, the expression of PDGFRα and PDGFRβ in DOCA group was increased on day 14. Compared with DOCA group, the expression of PDGFRα and PDGFRβ in DOCA+IMA group was attenuated. Compared with CON group, the expression of PDGFRβ, p-PDGFRβ, FSP-1, α-SMA, procollagenⅠand procollagen Ⅲ in DOCA group was increased significantly on day 28, but the expression of PDGFRα was not increased significantly, and the expression of PDGFRβ was higher than PDGFRα. Compared with DOCA group, the expression of PDGFRβ, p-PDGFRβ, FSP-1, α-SMA, procollagenⅠand procollagen Ⅲ in DOCA+IMA group was attenuated on day 28. (3) The fibroblasts in the myocardial interstitium of CON group were mainly vimentin positive, but not α-SMA positive on day 28,while the number of α-SMA-positive spindle-shaped cells (myofibroblasts) increased significantly in DOCA group. Compared with DOCA group, the number of myofibroblasts in DOCA+IMA group was attenuated on day 28. (4) PDGFRα was located in fibroblasts and myofibroblasts but not in vascular smooth muscle cells (VSMCs). PDGFRβ was not only located in myofibroblasts but also in VSMCs. CONCLUSION:PDGFRα takes effect in the early phase of myocardial fibrosis in the DOCA/salt hypertensive rats, while PDGFRβ takes effect in the entire process. PDGFRα and PDGFRβ are expressed in cardiac fibroblasts and myofibroblasts. Imatinib reduces the proliferation and transformation of fibroblasts, thereby attenuating myocardial fibrosis by inhibiting PDGFs/PDGFRs signaling pathway.