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

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

Effect of 27nt-miRNA on regulation of SM22α expression in vascular smooth muscle cells and its effect on cell viability,migration and phenotypic changes

AIM:To investigate the effect of 27nt-microRNA (27nt-miRNA) on the expression of smooth muscle 22α protein (SM22α) and the cell viability, migration and phenotypic changes of vascular smooth muscle cells (VSMCs). METHODS:The highly expression plasmids of 27nt-miRNA, and anti-27nt-miRNA and negative control plasmids were constructed, packaged with lentivirus and transfected into the rat primary VSMCs. Platelet-derived growth factor BB (PDGF-BB) was added to induce VSMCs phenotype conversion. The cell viability was measured by MTT assay. The migration ability was detected by scratch assay. The mRNA and protein expression of SM22α was determined by RT-PCR, immunocytochemical staining and Western blot. RESULTS:Compared with normal group, the cell viability in PDGF-BB group was increased (P<0.05), the migration ability was increased (P<0.05) and the expression of SM22α at mRNA and protein level was decreased (P<0.05). Compared with negative control lentiviral group, the cell viability in 27nt-miRNA over-expression group was decreased (P<0.05), the migration ability was decreased (P<0.05), and the mRNA and protein expression of SM22α was increased (P<0.05). While in anti-27nt-miRNA group, the cell viability was increased(P<0.05), the migration ability was increased (P<0.05), and the mRNA and protein expression of SM22α was decreased (P<0.05). CONCLUSION:27nt-miRNA significantly increases the expression of SM22α, while inhibits the viability and migration ability of VSMCs, and inhibits its phenotypic shift from contractile to synthetic.     

Effect of curcumin derivative B06 on liver from type 2 diabetic rats

AIM:To observe the protective effect of curcumin derivative B06 on the liver from the rats with hyperlipidemia and type 2 diabetes mellitus. METHODS:Male Sprague-Dawley rats (n=35) were divided randomly into 5 groups: normal control group, high-fat group, high-fat+B06-treated group, diabetic group and diabetic +B06-treated group. After fed with a high-fat diet for 4 weeks, the rats in the later 2 groups were injected with streptozotocin intraperitoneally to induce type 2 diabetes mellitus. The rats in B06-treated groups were given B06 by gavage at a dose of 0.2 mg· kg-1·d-1 for 8 weeks. After the treatment, the morphology of the liver was observed under light and transmission electron microscopes. The protein expression of AMP-activated protein kinase α (AMPKα) and phosphorylated AMPK α (p-AMPKα) was detected by Western blotting. RESULTS:Fatty degeneration, hepatocellular necrosis, inflammatory cell infiltration and hyperplasia of fibrous tissue were observed in the liver from the rats in high-fat group and diabetic group,and were relieved after B06 treatment. The protein expression of p-AMPKα was decreased in the liver of the rats in diabetic group and high-fat group, and it was increased in the liver of the high-fat and diabetic rats in B06-treated group. CONCLUSION:Curcumin derivative B06 exerts a protective effect on the liver in type 2 diabetic rats, and the increased expression of p-AMPKα may be involved in the mechanism of protection.     

Inhibition of plasminogen activator inhibitor-1 by small interfering RNA attenuates pulmonary fibrosis in rats

AIM: To examine the effects of silencing of plasminogen activator inhibitor-1 (PAI-1) expression by small interfering RNA (siRNA) on bleomycin (BLM)-induced rat pulmonary fibrosis. METHODS: Total 72 Wistar rats were divided into 4 groups: control, BLM, BLM+non-specific siRNA (BLM+N), and BLM+ PAI-1 siRNA (BLM+P). Pulmonary fibrosis was induced by intratracheal injection of BLM (5 mg/kg), whereas equal volume of normal saline was used in control group. After the administration of BLM or normal saline, the rats were treated with tracheal injection of PAI-1-siRNA (7.5 nmol/0.2 mL per rat) in BLM+P group, non-specific siRNA (7.5 nmol/0.2 mL per rat) in BLM+N group, and 0.2 mL normal saline in BLM group and control group, twice a week, 8 times in 28 d. On day 7, 14, and 28, the rats (n=6 at each time point) were sacrificed. The bronchoalveolar lavage fluid (BALF) from the left lung was harvested to examine the activity of PAI-1. The mRNA expression of collagen type Ⅲ, α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the middle lobe of the right lung was detected by RT-PCR. RESULTS: PAI-1 activity and the expression of collagen type Ⅲ, α-SMA and TIMP-1 were increased in BLM group on day 7, 14 and 28. Intratracheal injection of PAI-1 siRNA twice a week continuously reduced PAI-1 activity in the BALF (P<0.05),and decreased the expression of collagen type Ⅲ, α-SMA and TIMP-1 in the fibrotic lung tissues on day 7, 14 and 28. Statistical differences in the expression of collagen type Ⅲ, α-SMA and TIMP-1 between BLM+P group and BLM group at the same time point were observed. CONCLUSION: Intratracheal injection of PAI-1 siRNA twice a week continuously inhibits the expression of PAI-1. PAI-1 siRNA ameliorates BLM-induced pulmonary fibrosis by down-regulation of TIMP-1 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.     

Effects of astragalus injection combined with puerarin injection on process of endoplasmic reticulum stress through PERK pathway in dabe-tic nephropathy mice

AIM: To investigate the effects of astragalus injection combined with puerarin injection on endoplasmic reticulum stress through PERK pathway in diabetic nephropathy mice. METHODS: Male KKA^y mice were randomly divided into model group (injected with normal saline) and treatment group (injected with astragalus and puerarin). The male C57BL/6J mice served as normal group. The mice were sacrificed 4 weeks after treatments for observing morphological changes under electron microscope. The renal tissues were collected to determine the expression of protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2α (eIF2α) and glucose-regulated protein 78 (GRP78) at mRNA and protein levels by real-time PCR and Western blot. RESULTS: Under electron microscope, the renal tubular epithelial cells in model group and treatment group showed the swelling of the nucleus, endoplasmic reticulum and mitochondria. The results of real-time PCR and Western blot showed that the expression of PERK, eIF2α and GRP78 at mRNA and protein levels in model group was higher than that in normal group (P<0.05), while that in treatment group was lower than that in model group. CONCLUSION: Astragalus injection combined with puerarin injection reduces the mRNA and protein expression of PERK, eIF2α and GRP78, thus inhibiting the endoplasmic reticulum stress in type 2 diabetic mice to protect the kidney function.     

Effects of extract of gingko biloba on expression of glucocorticoid receptor in hepatic tissues from type 2 diabetic rats

AIM: To study the effect and mechanism of extract of ginkgo biloba (EGB) on liver glucocorticoid receptor (GR) expression in type 2 diabetic rats. METHODS: Thirty male Sprague-Dawley rats were divided randomly into three groups: normal control group (n=10), type 2 diabetic group (n=10) and ginkgo biloba treated group (n=10). After fed with high-fat feeding for 4 weeks, the later two groups were injected with streptozotocin at a dose of 30 mg/kg intraperitoneally to induce type 2 diabetic rat model. The EGB treated group was gavaged with EGB at the dose of 50 mg·kg-1·d-1 for 12 weeks. At the end of experiment, the rats were sacrificed, the blood glucose, serum lipid and blood insulin were measured. The morphology of liver tissue was observed under light microscopy with HE staining. GR mRNA expression in liver was measured by RT-PCR. The level of GR protein expression in liver tissue was detected by immunohistochemistry. RESULTS: EGB reduced the levels of blood glucose, blood lipids, blood insulin in diabetic rats. EGB also relieved fatty degeneration and necrosis of the hepatic cells, ameliorated infiltration of inflammatory cells in the liver; and decreased GR expression at both mRNA and protein levels in diabetic liver. CONCLUSION: EGB may inhibit GR expression in liver of type 2 diabetic rats, which results in decreasing the level of blood glucose, blood lipid, blood insulin and relieving the liver damage in type diabetic rats.     

Effects of epididymal P34H gene silencing on expression of P34H and activity of hyaluronidase in mouse sperm

AIM: To investigate the effects of P34H gene silencing on the expression of P34H and activity of hyaluronidase (HYD) in mouse sperm.METHODS: The recombinant plasmid series of P34H targeted short hairpin RNA (shRNA) were constructed by GV248 plasmids vector. These recombinant plasmids were transformed into DH5α competent cells, and the plasmids were taken from DNA sequencing analysis. The HEK293T cells were co-transfected with shRNA and lentiviral packaging plasmids. The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to inject into the mouse epididymis and the expression of P34H at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The location of P34H protein on the mouse spermatozoa was determined by indirect immunofluorescent staining using P34H antibody. The positive rate and activity intensity of HYD was detected by modified sodium hyaluronate-gelatin membrane. RESULTS: DNA sequencing analysis confirmed that the 3 P34H-shRNA sequences were successfully inserted into the lentiviral vectors. P34H expression in epididymis tissue was significantly decreased at both mRNA and protein levels compared with those of the non-transfected and normal control vectors (P<0.05). The GV-P34H-shRNA-1 played a significant role in reducing the percentage of P34H positive rate and the activity of HYD in mouse sperm. The silencing effect did not significantly differ between the non-transfected and normal control vectors. CONCLUSION: Silencing of P34H significantly inhibits the percentage of P34H positive rate and the activity of hyaluronidase in mouse sperm.     

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.     

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.     

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.     

Effect of circRNA_000203 on fibrotic phenotypes in mouse cardiac fibroblasts

AIM: To determine circular RNA (circRNA) profiles in the diabetic mouse myocardium, and to investigate the effect of circRNA_000203 on fibrotic phenotypes in cardiac fibroblasts.METHODS: Masson trichrome staining was performed on the myocardium of the diabetic db/db mice and the non diabetic db/m control mice. circRNA expression profile in the diabetic myocardium was detected by circRNAs microarray. The expression of circRNA_000203 was determined by real time fluorescence quantitative PCR (RT-qPCR). Recombinant circRNA_000203 adenovirus was prepared for enforced the expression of circRNA_000203 in mouse cardiac fibroblasts. The expression of Col1a2, Col3a1and α-SMA was determined in circRNA_000203-modified cardiac fibroblasts, respectively. RESULTS: Masson trichrome staining showed that fibrosis was increased in the diabetic mouse myocardium. The results of circRNA array detection revealed that circRNAs were dysregulated in the diabetic myocardium. circRNA_000203 was up-regulated in the diabetic myocardium. Significant over-expression of circRNA_000203 was achieved in the cardiac fibroblasts after infection with the recombinant circRNA_000203 adenovirus. The mRNA and protein expression of Col1a2, Col3a1 and α-SMA was significantly increased in the cardiac fibroblasts with over-expression of circRNA_000203.CONCLUSION: circRNA_000203 is up-regulated in the diabetic mouse myocardium. It has pro-fibrotic effect on the cardiac fibroblasts.     

Effect of cyclopamine on aristolochic acid-induced phenotypic transformation and Hedgehog pathway in renal epithelial cells

AIM: To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transformation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E. METHODS: NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at concentrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10 μmol/L). After cultured for 24 h, the mRNA expression of Ptch1, Smo, α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR. The protein levels of Shh and TGF-β1 were detected by ELISA. Immunofluorescence staining was used to evaluate the expression of Ptch1, Smo, α-SMA, E-cadherin and type III collagen in the NRK-52E cells. RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling, and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells. Treatment with cyclopamine inhibited HH signaling by decreasing Smo expression and increasing Ptch1 expression. Moreover, cyclopamine also down-regulated the expression of TGF-β1, α-SMA, type I collagen and III collagen, and up-regulated the expression of BMP-7, ZO-1 and E-cadherin. CONCLUSION: AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells, which can be inhibited by cyclopamine treatment. The possible mechanism is that cyclopamine suppresses the activation of HH signaling, resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.     

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.     

Inhibitory effect of oxymatrine on high glucose-induced rat renal tubular epithelial-mesenchymal transition

AIM:To investigate the inhibitory effect of oxymatrine (OM) on high glucose-induced rat renal tubular epithelial-mesenchymal transition (EMT). METHODS:The rat renal tubular epithelial NRK52E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+different concentrations of OM groups and high glucose+0.50 g/L OM dynamic observation group. The expression of TGF-β1, Smad7, α-SMA and E-cadherin at mRNA and protein levels was detected by real-time PCR and Western blotting. The viability of NRK52E cells was determined by MTT assay. RESULTS:(1) Compared with control group, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose group gradually increased, and Smad7 protein and E-cadherin mRNA and protein gradually reduced, but the mRNA expression of Smad7 gradually increased. (2) Compared with high glucose group, as increases in OM doses, the expression of TGF-β1 and α-SMA at mRNA and protein levels in high glucose+different concentrations of OM groups gradually reduced, and Smad7 protein and E-cadherin mRNA and protein gradually increased, but the mRNA expression of Smad7 had no significant change. (3) Compared with high glucose group, the expression of TGF-β1 and α-SMA at mRNA and protein levels was significantly reduced, the expression of E-cadherin at mRNA and protein levels significantly increased, and the protein expression of Smad7 significantly increased, but the mRNA expression of Smad7 had no significant change in high glucose+0.50 g/L OM dynamic observation group. CONCLUSION: In NRK52E cells, oxymatrine inhibits high glucose induced EMT by down-regulating TGF-β1 and up-regulating Smad7, thus preventing the fibrosis effect of TGF-β1/Smads signaling.     

Endogenous nitric oxide synthase inhibitor increase skeletal muscle contractility and mitochondria biosynthesis in 4-week running rats

AIM: To observe the effect of endogenous nitric oxide synthase(NOS) inhibitor asymmetric dimethylarginine(ADMA) and its signaling pathways on NO levels and skeletal muscle contractility in 4-week running rats. METHODS: The 4 weeks running rat model was established. The twitch tension, tetanic tension and the fatigue test of soleus muscle induced by electrical stimulation ex vivo were detected. The ATP content, mitochondrial DNA levels and the mRNA expression of peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α), nuclear respiratory factor(NRF) were measured to reflect the mitochondrial function and biosynthesis in the skeletal muscle. Serum ADMA concentration was detected by high performance liquid chromatography. The endogenous ADMA enzymes PRMT1 and 2 subtypes of ADMA metabolism enzyme DDAH, 3 subtypes of NOS protein expression in the skeletal muscle were determined by Western blot. NOS activity and nitric oxide(NO) content were analyzed by colorimetric method. RESULTS: Compared with normal control group, the twitch tension, tetanic tension and the anti-fatigue capability of soleus muscle in running group were significantly enhanced, ATP content, mitochondrial DNA content and the mRNA expression of PGC-1α and NRF were significantly increased(P<0.01). In addition, the protein expression of constitute type NOS(cNOS) and NOS activity were significantly increased(P<0.01), but the increase in NO content was relatively smaller in soleus muscle in exercise group(P<0.05). Moreover, serum ADMA concentration in running group was increased, while the DDAH2 expression in skeletal muscle was decreased.CONCLUSION: Short-term endurance exercise enhances the twitch tension, tetanic tension and fatigue resistance of soleus muscle. The mechanism may be that increased cNOS expression feedbacks to increase ADMA concentration, thus maintaining the increase in NO synthesis at a relatively low level, and resulting in promoting skeletal muscle mitochondria biosynthesis and mitochondrial function.     

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。     

α2-adrenoceptor agonist B-HT933 suppresses LPS-induced TNF-α production in neonatal rat cardiomyocytes

AIM: To observe the effect of B-HT933, a selective α₂-adrenoceptor agonist, on lipopolysaccharide(LPS)-induced TNF-α production in neonatal rat cardiomyocytes and to explore the underlying mechanisms.METHODS: The neonatal rat cardiomyocytes were cultured. The localization of α_2A-adrenoceptor in the cardiomyocytes was examined by immunofluorescence staining. The cardiomyocytes were exposed to LPS or/and B-HT933 for different time. The level of TNF-α in the supernatants and the mRNA expression of TNF-α were detected by ELISA and real-time PCR, respectively. In addition, LPS-associated signal molecules in the cardiomyocytes were also examined by Western blotting.RESULTS: Immunofluorescence staining showed that α_2A-adrenoceptors were localized in the cardiomyocytes. LPS stimulated TNF-α production in the cardiomyocytes in a dose and time-dependent manner. B-HT933 pretreatment significantly inhibited the expression of TNF-α at mRNA and protein levels in LPS-treated cardiomyocytes. Furthermore, LPS exposure induced IκBα and p38 phosphorylation in cardiomyocytes and only IκBα phosphorylation was prevented by B-HT933 treatment.CONCLUSION: α_2A-adrenoceptors are present in neonatal rat cardiomyocytes and its agonist B-HT933 inhibits LPS-induced TNF-α production in cardiomyocytes via suppressing IκBα phosphorylation.