Effect of miR-29b-mediated TGF-β/Smad signaling pathway on progression of hepatic fibrosis in rats

AIM: To investigate the role of microRNA-29b (miR-29b)-mediated TGF-β/Smad signaling pathway in the activation of hepatic stellate cells (HSC) and its effect on the progression of hepatic fibrosis in rats.METHODS: Hepatic liver fibrosis rat model was established, and its HSC were isolated. Normal rat HSC were also obtained and identified in vitro. RT-qPCR and Western blot were used to detect the alterations of miR-29b, TGF-β/Smad signaling pathway-related proteins and liver fibrosis marker proteins in the acquired cells. Finally, the direct targeting binding of miR-29b to TGF-β1 was identified by dual-luciferase reporter assay system.RESULTS: With the activation of HSC, the expression of miR-29b gradually decreased (P<0.01), while the expression of collagen type I and α-smooth muscle actin gradually increased (P<0.01). At the same time, the expression of Smad2/3/4 was significantly increased, and the expression of Smad7 was significantly decreased (P<0.01). Dual-luciferase reporter assay showed that miR-29b bound directly to "UCUCUCCGU" in the 3'UTR of TGF-β1, indicating that TGF-β1 was a downstream target gene of miR-29b.CONCLUSION: miR-29b may be involved in the inhibition of HSC activation and migration, thereby inhibiting the process of liver fibrosis. The biological function of miR-29b may be through the direct targeting of TGF-β1, thus regulating and inhibiting the TGF-β/Smad signaling pathway.     

Role of EndoMT in HHPH based on TGF-β/Smads signaling pathway and regulated by Yiqi-Wenyang-Huoxue-Huatan formula

AIM: To investigate the relationship between transforming growth factor-β (TGF-β)/Smads signaling pathway and pulmonary arterial endothelial-mesenchymal transition (EndoMT) in hypoxia-hypercapnia pulmonary hypertension (HHPH) process and the regulatory effect of Yiqi-Wenyang-Huoxue-Huatan formula (YWHHF). METHODS: Healthy male SD rats were randomly divided into 5 groups:normal control (N) group, hypoxia-hypercapnia (HH) group, high-dose YWHHF (YH) group, middle-dose YWHHF (YM) group and low-dose YWHHF (YL) group. The rats in N group was housed in normoxic environment, and the rats in the other 4 groups were housed in hypoxia-hypercapnia environment (9%~11% O₂ and 5%~6% CO₂) for 4 weeks, 8 h/d, 6 d/week. The excess water vapor was absorbed by anhydrous CaCl₂, and CO₂ was absorbed by sodium hydroxide. The rats in YWHHF groups were put into the oxygen chamber before the same volume of YWHHF at different concentrations were given (200 g/L for YH group, 100 g/L for YM group and 50 g/L for YL group). The average pulmonary artery pressure and the average carotid artery pressure were measured during the operation. After operation, the right ventricular free wall and left ventricle plus interventricular septum were collected for determining the right ventricular hypertrophy index. Moreover, the morphological changes of the lung tissues were observed under light microscope. The mRNA and protein levels of α-smooth muscle actin (α-SMA), CD31, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were detected by RT-PCR and Western blot. RESULTS: Compared with N group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were increased, the levels of CD31 were decreased (P<0.05), and the lung tissue damage was observed in the other 4 groups. Compared with HH group, the pulmonary artery mean pressure, the mRNA and protein expression of α-SMA, TGF-β1 and Smad2/3, and the protein level of p-Smad2/3 were decreased, while the mRNA and protein levels of CD31 were increased. Moreover, the lung tissue damage was reduced in YH, YM and YL groups. CONCLUSION: TGF-β/Smads pathway may be involved in the process of EndoMT under hypoxia and hypercapnia condition, and YWHHF may reduce EndoMT by inhibiting the expression of TGF-β/Smads pathway-related molecules.     

Effect of alpha-lipoic acid on expression of miR-21/Smad7 and fibrosis in kidney of diabetic rats

AIM: To investigate the protective effect of alpha-lipoic acid (ALA) on the kidney of the rats with diabetes mellitus (DM), and to discuss the mechanism. METHODS: The DM rats were divided into normal control (NC) group, DM group and ALA group. After treated with ALA for 6 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and the pathological changes of the kidney tissues were observed by HE staining and Masson staining. The protein levels of transforming growth factor-β1 (TGF-1), p-Smad2/3, Smad7, collagen I and collagen Ⅲ were determined by Western blot. In addition, the expression of microRNA-21 (miR-21) was detected by RT-qPCR. RESULTS: Compared with NC group, the kidney weight/body weight, blood glucose (BG), total cholesterol, triglyceride and 24-h urine protein were remarkably increased in DM group (P<0.05). The pathological observation of the kidney tissues showed fibrosis changes in DM group. The level of Smad7 was reduced in DM group, while the levels of TGF-β1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were increased (P<0.05). After treatment with ALA for 6 weeks, all the relevant biochemical parameters were reduced except BG, and the renal fibrosis lesions were obviously alleviated. Compared with DM group, the levels of TGF-1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were reduced in ALA group, while the level of Smad7 was increased (P<0.05).CONCLUSION: ALA may prevent the development of renal fibrosis in rats through restraining the expression of TGF-β1 and miR-21, increasing the levels of Smad7 protein, and reducing the deposition of extra cellular matrix.     

Effects of Danshensu on TGF-β1/Smads signaling pathways in rats with pulmonary fibrosis

AIM: To study the preventive and curative roles of Danshensu (DA) in bleomycin (BLM)-induced pulmonary fibrosis in rats. METHODS: Pulmonary fibrosis was induced in SD rats by intratracheal instillation of BLM. The rats were intraperitoneally injected with dexamethasone (1 mg·kg-1·d-1, DXM group), DA (15 mg·kg-1·d-1, DA group), or physiological saline (2 mL·d-1, BLM group). Normal controls (NC group) received physiological saline both intratracheally and intraperitoneally. At the 28th day after modeling, the histological changes of the lungs were evaluated by hematoxylin-eosin (HE) and Masson’s trichrome staining. The protein levels of α-smooth muscle actin (α-SMA) in the lung tissues were detected by the method of immunohistochemistry. The mRNA expression of transforming growth factor beta 1 (TGF-β1), Smad3 and Smad7 was assessed by real-time fluorescence quantitative PCR. RESULTS: Compared with BLM group, the degree of inflammation and fibrosis of the lung in DA group was obviously reduced, and so was the expression of α-SMA in the lung tissues. The mRNA expression of TGF-β1 and Smad3 in the lung tissues of the rats decreased and the mRNA expression of Smad7 increased. CONCLUSION: DA alleviates BLM-induced pulmonary fibrosis in rats in the early stage by inhibiting the expression of TGF-β1/Smad3 and stimulating the expression of Smad7 in the lung tissues.     

Role of hepatitis B virus in intrahepatic TGF-β1/Smads signaling pathway

AIM:To explore the effects of hepatitis B virus (HBV) on intrahepatic expression of transforming growth factor β1（TGF-β1） and Smads. METHODS:The expression of intrahepatic TGF-β1, HBsAg and HBcAg in control group and chronic hepatitis B (CHB) group was detected by immunohistochemical method.The serum HBV DNA content was determined by real-time PCR. The role of HBV in the expression of TGF-β1, Smad3 and Smad7 in human hepatic stellate cell line LX-2 in vitro was observed by cell culture and Western blotting. RESULTS:The average score of intrahepatic TGF-β1 expression in CHB group was higher than that in control group. With the increase in serum HBV DNA content, intrahepatic TGF-β1 expression was also enhanced. In the HBcAg positive hepatic tissue, there was higher TGF-β1 expression than that in the liver tissue of HBcAg negative. Compared with control group and HBV+anti-TGF-β1 group, HBV caused increased expression of TGF-β1 and Smad3 in HBV group in vitro. No difference of Smad7 protein among control group, HBV group and HBV+anti-TGF-β1 group was observed. CONCLUSION: The expression of intrahepatic TGF-β1 is related to serum HBV DNA and hepatocellular HBcAg in the patients with CHB. HBV-induced liver fibrosis mainly relies on positive regulatory mechanisms of Smad3,and the negative regulation by Smad7 almost does not function.     

Effect of rhynchophylline on TGF-β1/Smad pathway for processing ventricular remodeling in spontaneously hypertensive rats

AIM: To investigate the effect of rhynchophylline (Rhy) on blood pressure, cardiac hypertrophy and myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Spontaneously hypertensive rats were randomly divided into model group, high dose (10 mg·kg^-1·d^-1) and low dose (2.5 mg·kg^-1·d^-1) group of rhynchophylline, captopril group (17.5 mg·kg^-1·d^-1). Wistar-Kyoto rats were used as normal control. Respectively, systolic blood pressure was measured by tail cuff every 2 weeks. After 10 weeks, heart weight index and left ventricular weight index were calculated. The myocardial hydroxyproline and plasma angiotensin Ⅱ were detected. Moreover, basic myocardial histopathological changes and myocardial collagen fibres were observed by HE staining and Masson staining, respectively. The protein expression of TGF-β₁ and Smad3 in the myocardium was measured by the methods of immunohistochemistry and Western blot. RESULTS: Compared with SHR model group, Rhy significantly reduced blood pressure (P<0.05), the levels of HYP in the myocardium (P<0.05) and the levels of AngⅡ in the plasma (P<0.01). The pathological damages of the myocardial tissues and collagen deposition were attenuated. The protein expression of TGF-β₁ and Smad3 was significantly reduced by the treatment with Rhy (P<0.01). CONCLUSION: Rhynchophylline reduces blood pressure and adjusts to improve ventricular remodeling of SHR. The mechanism may be involved in the TGF-β₁/Smad pathway and reducing AngⅡ content.     

Lentiviral vector CV072-mediated Mfn2 over-expression inhibits hepatic stellate cell activation

AIM:To construct a lentiviral vector carrying mitofusin 2 (Mfn2), and to investigate the inhibitory effect of Mfn2 on the activation of rat hepatic stellate cells and its mechanism of reducing the formation of hepatic fibrosis-related factors. METHODS:The lentiviral over-expression vector CV072-pCMV-Mfn2-EGFP containing Mfn2 was constructed and transfected into the hepatic stellate cells. The expression of green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was evaluated. The protein levels of Bax, Bcl-2, cleaved caspase-3, α-SMA, TGF-β1, Smad2 and Smad3 were detected by Western blot. The levels of type I collagen, type Ⅲ collagen and type IV collagen in the cell culture supernatants were determined by ELISA. RESULTS:Compared with control group, the apoptosis of the hepatic stellate cells transfected with lentivirus over-expression vector CV072-pCMV-Mfn2-EGFP was increased, and the protein levels of proapoptotic molecules Bax and cleaved caspase-3 were increased (P<0.01). TGF-β1/Smad pathway-related proteins TGF-β1, p-Smad2 and p-Smad3 were decreased, and the levels of fibrosis-related proteins α-SMA, type I collagen, type Ⅲ collagen and type IV collagen were decreased (P<0.01). CONCLUSION:Transfection of lentiviral over-expression vector CV072-pCMV-Mfn2-EGFP effectively inhibits hepatic stellate cell activation in vitro and may reduce the production of hepatic fibrosis-related factors by inhibiting TGF-β1/Smad pathway.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Effects of PI3K/PKB signaling pathway on expression of osteopontin in human hepatic stellate cells induced by transforming growth factor-β1

AIM: To investigate the regulatory effects of phosphatylinositol 3-kinase/protein kinase B (PI3K/PKB) signaling pathway on the expression of osteopontin (OPN) in transforming growth factor-β₁ (TGF-β₁)-induced human hepatic stellate cells. METHODS: Human hepatic stellate cell line LX-2 was cultured in DMEM and stimulated by TGF-β₁ at the final concentration of 2.5, 5, 10 and 20 μg/L for 24 h or at final concentration of 10 μg/L for 12 h, 24 h and 48 h. LX-2 cells were pretreated with wortmannin, a specific inhibitor of PI3K/PKB signaling pathway, at final concentration of 0.1 μmol/L for 1 h, followed by incubation with TGF-β₁ at final concentration of 10 μg/L for 24 h. The cells were collected. The expression of OPN was detected by real-time PCR and Western blotting. RESULTS: In LX-2 cells, the expression of OPN was apparently elevated when incubated with TGF-β₁. With the increase in TGF-β₁ concentration or the extension of incubation hours, the expression of OPN was increased gradually in a dose-and time-dependent manner with certain limits. LX-2 cells pretreated with wortmannin and incubated with TGF-β₁ had a significant decrease in the OPN expression as compared with control group (P<0.01). CONCLUSION: The expression of OPN in TGF-β₁-induced LX-2 cells is regulated by the PI3K/PKB signaling pathway.     

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

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

Role of BMP-7/Smads pathway in regulation of EndoMT in rats with HHPH

AIM: To investigate the role of bone morphogenetic protein 7 (BMP-7)/Smads pathway in the re-gulation of endothelial-mesenchymal transition (EndoMT) in rats with hypoxia-hypercapnia pulmonary hypertension (HHPH). METHODS: The rat pulmonary artery endothelial cells (RPAECs) were divided into normoxic control (Con) group, hypoxia-hypercapnia (HH) group, BMP receptor agonist rhBMP-7 group, BMP receptor inhibitor DMH-1 group and solvent DMSO group. After given the corresponding drugs in each group, the cells in Con group were cultured in a normal-oxygen incubator, and the cells in the remaining 4 groups were cultured in a low-oxygen and high-carbon-dioxide incubator. The expression levels of CD31 and α-smooth muscle actin (α-SMA) were observed by immunofluorescence staining. The mRNA and protein expression levels of α-SMA, CD31 and Smad1/5/8 were detected by RT-PCR and Western blot, respectively. The cell viability was measured by CCK-8 assay. The cell migration ability was detected by Transwell chamber assay. RESULTS: Compared with Con group, the expression of α-SMA at mRNA and protein levels in HH group was increased, the expression levels of CD31 mRNA and protein, Smad1/5/8 mRNA and p-Smad1/5/8 protein were decreased, the cell viability was decreased and the number of migratory cells was increased (P<0.05). Compared with HH group, the expression of α-SMA at mRNA and protein levels in rhBMP-7 group was decreased, the expression levels of CD31 mRNA and protein, Smad1/5/8 mRNA and p-Smad1/5/8 protein were increased, the cell viability was increased and the number of migratory cells was reduced (P<0.05). Compared with rhBMP-7 group, the expression of α-SMA at mRNA and protein in DMH-1 group was increased, the expression levels of CD31 mRNA and protein, Smad1/5/8 mRNA and p-Smad1/5/8 protein were decreased, the cell viability was decreased and the number of migratory cells was increased (P<0.05). CONCLUSION: Hypoxia-hypercapnia conditions promote EndoMT in RPAECs, which promotes the development of hypoxia-hypercapnia pulmonary hypertension, and the underling mechanism may be related to the inhibition of BMP-7/Smads pathway.     

Effect of Shaofu-Zhuyu decoction on MAPK/ERK signaling pathway in rats with endometriosis

AIM: To observe the effects of Shaofu-Zhuyu decoction (SFZY) on mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway in the rats with endometriosis (EM), and to explore the mechanism of SFZY for treatment of EM.METHODS: Healthy female SD rats were used to establish the EM model. The rats were randomly divided into blank control group, model group, positive control group, and low dose, middle dose and high dose of SFZY groups. The pathological changes of the endometriotic tissue were observed by HE staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-6(IL-6) and IL-8 in the uterine tissue were detected by ELISA. The mRNA expression of ERK, vascular endothelial growh factor (VEGF) and matrix metalloprotein-9 (MMP-9) was detected by RT-qPCR. The protein expression of nuclear factor-κB (NF-κB), MAPK and MAPK-ERK kinase (MEK) was determined by Western blot.RESULTS: Compared with model group, the levels of TNF-α, IL-6 and IL-8 in the uterine tissue of the rats in middle dose and high dose of SFZY groups were significantly decreased (P<0.05), the mRNA expression of ERK, VEGF and MMP-9 was significantly reduced, and the protein expression of NF-κB, MEK and MAPK was decreased significantly in the rat endometriotic tissues (P<0.05).CONCLUSION: SFZY may play a key role in the treatment of EM by regulating MAPK/ERK signaling pathway.     

Effect of EZH2 regulating Wnt/β-catenin signaling pathway on apoptosis of brain glioma cells

AIM: To investigate the effect of enhancer of zeste homolog 2 (EZH2) regulating Wnt/β-catenin signaling pathway on the apoptosis of brain glioma cell lines. METHODS: The expression level of EZH2 in glioma cell lines U87, H4 and U251 and normal human astrocytes (NHA) was detected by RT-qPCR and Western blot. The EZH2 siRNA and siRNA control were transfected into the H4 cells. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Caspase-3 activity was detected by spectrophotometry. The expression levels of the key protein β-catenin of the Wnt/β-catenin signaling pathway and the downstream target molecule c-Myc were determined by Western blot. After the H4 cells transfected with EZH2 siRNA were treated with an activator of Wnt/β-catenin signaling pathway, the apoptosis rate was measured by flow cytometry, and the expression of β-catenin and c-Myc was determined by Western blot. RESULTS: The mRNA and protein expression levels of EZH2 in the glioma cell lines U87, H4 and U251 were significantly higher than those in NHA (P<0.05). The expression of EZH2 at mRNA and protein levels in the H4 cells was higher than that in U87 cells and U251 cells (P<0.05). EZH2 siRNA obviously inhibited the expression of EZH2 at mRNA and protein levels in the H4 cells. Knockdown of EZH2 expression decreased the viability of H4 cells, the apoptotic rate was significantly increased, and the activity of caspase-3 was significantly increased in the cells (P<0.05). Knockdown of EZH2 expression also inhibited the expression of β-catenin and c-Myc. The activator of Wnt/β-catenin signaling pathway reduced the apoptosis rate of H4 cells induced by down-regulation of EZH2, and reduced the activity of caspase-3 in the cells. CONCLUSION: EZH2 is over-expressed in glioma cells. Down-regulation of EZH2 expression induces apoptosis of glioma cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Effect of bFGF on promoting angiogenesis in infarct area and improving myocardial fibrosis in mouse myocardial infarction model

AIM: To investigate the protective effect of basic fibroblast growth factor (bFGF) on the heart of mice with myocardial infarction and its mechanism. METHODS: The model of myocardial infarction was established by the ligation of left anterior descending artery of C57/B6 mice (8~12 weeks old) after lateral thoracotomy. The mice were divided into sham operation group, myocardial infarction group and bFGF administration group. bFGF at 0.5 μg was intraperitoneally injected on alternate days after myocardial infarction for 7 d. Cardiac Doppler ultrasonography was used to detect cardiac function after myocardial infarction for 28 d, and left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction and left ventricular shortening fraction (LVFS) were used to evaluate cardiac function. After myocardial infarction for 28 d, the mice were sacrificed and the hearts were collected for preparing pathological sections. The degrees of myocardial fibrosis and angiogenesis in the myocardial infarction area were observed. Western blot was used to detect the indicators of angiogenesis. RESULTS: The results of Masson staining showed that bFGF administration significantly reduced myocardial fibrosis at 28 d after myocardial infarction. Cardiac ultrasound data showed that cardiac functions in myocardial infarction group were poorer than those in sham group, and bFGF administration significantly improved cardiac functions. Immunofluorescence staining showed that neovascularization in myocardial infarction area of bFGF administration group was more than that in myocardial infarction group. The results of Western blot showed that bFGF activated AKT/HIF-1α/VEGF signaling pathway. CONCLUSION: Intraperitoneal injection of bFGF reduces myocardial fibrosis and improves cardiac function in myocardial infarction mice. bFGF may promote angiogenesis by activating AKT/HIF-1α/VEGF signaling pathway.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Effects of piceatannol on rat kidney with diabetic nephropathy in early stage

AIM: To observe the effect of piceatannol on the kidney of diabetic nephropathy rats in early stage, and to explore the possible mechanisms.METHODS: The rats were randomly divided into 5 groups:control group, model group, low dose of piceatannol treatment group, medium dose of piceatannol treatment group and high dose of piceatannol treatment group. The rat model of diabetic nephropathy was induced accordingly, and the rats received 20 mg/kg, 40 mg/kg or 60 mg/kg of piceatannol by gavage once a day for 4 weeks. Blood glucose was detected by glucometer. The urea nitrogen and creatinine levels in the serum were measured by urease-glutamate dehydrogenase enzymatic and inosine acid oxidase methods, respectively, and 24 h urinary microalbumin was analyzed by immune transmission turbidimetry test. Moreover, the pathological changes of the kidney tissues were observed under microscope with HE staining. The protein expression of TGF-β1 and Smad 7 and the phosphorylation levels of Smad2 and Smad3 were determined by Western blot. RESULTS: Compared with model group, piceatannol treatment significantly decreased the levels of blood glucose, blood urea nitrogen and urinary microalbumin, but had no effects on serum creatinine. Furthermore, HE staining showed that the increased mesangial cells, matrix hyperplasia and degenerated epithelial cells in model group were markedly inhibited after piceatannol treatment. Additionally, piceatannol treatment also reduced the protein expression of TGF-β1 and Smad 7, and the phosphorylation levels of Smad2 and Smad3. CONCLUSION: Piceatannol attenuates pathological progression in the kidney of diabetic nephropathy rats in early stage, which may be through inhibiting TGF-β/Smad signaling pathway.     

Emodin reduces hypoglycemia/hypoxia-induced injury of microglia by TLR4/NF-κB signaling pathway

AIM: To investigate the mechanism of emodin on the protection of glucose-deficient/anoxic microglia. METHODS: A microglia BV2 cell model induced by hypoglycemia/hypoxia (HH) was established. The glucose-deficient/anoxic cells treated with emodin were labeled as HH+emodin (20, 40 and 80 μmol/L) groups. The BV2 cells with TLR4 over-expression treated with emodin under hypoglycemia/hypoxia condition was labeled as HH+pcDNA-TLR4+ emodin (40 μmol/L) group. The cell viability was measured by MTT assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were detected by ELISA. The apoptosis was analyzed by flow cytometry. The protein levels of Bax, Bcl-2, TLR4, p-IκB and IκB were determined by Western blot. RESULTS: Compared with HH+DMSO group, the viability was significantly increased, the levels of LDH and TNF-α and apoptotic rate were significantly decreased, the protein levels of Bax, TLR4 and p-IκB were significantly decreased, the protein level of Bcl-2 was significantly increased in HH+emodin groups (P<0.05). Over-expression of TLR4 reversed the effect of emodin on promoting the viability and inhibiting apoptosis in the BV2 cells. CONCLUSION: Emodin has a protective effect on hypoglycemia/hypoxia induced microglia, and its mechanism may be related to the inactivation of TLR4/NF-κB signaling pathway.     

Rhein attenuates bleomycin-induced rats pulmonary fibrosis through TGF-β1/Smad pathway by inhibiting miR-21 expression

AIM: To investigate the effect of rhein on bleomycin-induced pulmonary fibrosis and the expression of microRNA-21 (miR-21) and transforming growth factor-β1 (TGF-β1)/Smad signaling molecules in rats. METHODS: A single dose of bleomycin was intratracheal injected into the SD rats to induce pulmonary fibrosis. After injection of bleomycin, the rats were randomly divided into low-, medium-and high-dose rhein treatment groups and model group. The rats that were instilled with normal saline intratracheally served as control group. After the treatment for 28 d, the pulmonary pathologic changes were observed under microscope with hematoxylin-eosin staining. The lung coefficient and hydroxyproline content were also measured. The expression of miR-21 and the mRNA levels of TGF-β1 and Smad7 in the lung tissues were detected by real-time PCR. The protein levels of TGF-β1 and Smad7 were determined by Western blot. RESULTS: Rhein significantly attenuated the experimental alveolitis, pulmonary fibrosis, lung coefficient and hydroxyproline contents in the rats. Rhein obviously decreased the expression of miR-21,and the mRNA and protein levels of TGF-β1, but significantly increased the mRNA and protein levels of Smad7 in the lung tissues. CONCLUSION: Rhein effectively prevents bleomycin-induced pulmonary fibrosis by inhibiting the expression of miR-21 and promoting the expression of Smad7, thus regulating the TGF/Smad signaling pathway to decrease extracellular matrix deposition.     

Notch1 promotes renal tubulointerstitial fibrosis in renal tissues of diabetic mice by inhibiting autophagy through Akt/mTOR signaling pathway

AIM: To observe the changes of Notch1 expression and autophagy in the renal tissues of diabetic mice, and to explore the regulatory effect of Notch1 on tubulointerstitial fibrosis by inhibiting autophagy in diabetic nephro-pathy. METHODS: The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice), with 8 rats in each group. After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. The protein expression of Notch1 in the renal tubular epithelial cells was observed by immunohistochemical staining. The protein levels of Notch1, PTEN, p-Akt (Thr308), Akt, p-mTOR (Ser2448), mTOR, LC3, P62, collagen type Ⅰ (Col-Ⅰ) and collagen type Ⅲ (Col-Ⅲ) were determined by Western blot. RESULTS: Compared with the db/m mice, the blood glucose, glycosylated hemoglobin, serum creatinine, triglyceride and total cholesterol were increased in the db/db mice (P<0.01). Renal tubular epithelial cell vacuolar degeneration, renal tubular expansion and interstitial inflammatory cell infiltration in db/db mouse renal tissues with HE staining were observed. The images of Masson staining showed collagenous fiber-like substance deposition in the glomerular capillaries and renal interstitium, and disarrangement of tubular structure in the renal tissues of db/db mice. The protein expression levels of PTEN and LC3-Ⅱ were decreased (P<0.01 or P<0.05), while the protein levels of Notch1, P62, p-mTOR (Ser2448), p-Akt (Thr308), Col-I and Col-III were increased in the db/db mice as compared with the db/m mice (P<0.01). However, no significant change of total mTOR and Akt proteins between the 2 groups was found. CONCLUSION: Notch1 protein expression was increased, PTEN expression was significantly reduced, Akt/mTOR pathway was activated, autophagy was inhibited, and fibrosis was aggravated in the renal tissues of the diabetic mice.