Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.     

The roles of ERK signaling pathway in osteogenic differentiation of rat MSCs promoted by quercetin

AIM:To study the roles of extracellular signal-regulated kinase(ERK) signal pathway in the process of osteogenic differentiation in rat mesenchymal stem cells(MSCs) promoted by quercetin(QUE). METHODS:The optimal concentration of QUE for promoting osteogenic differentiation of rat MSCs was determined by MTT and alkaline phosphatase(ALP) detection. The activity of ALP was detected by the ALP detection kit. The expression of bone Gla protein(BGP) and collagen typeⅠ(ColⅠ) was observed by ELISA analysis. MSCs were exposed to QUE at optimal concentration with or without ERK1/2 inhibitor PD98059. Non-phosphorylated and phosphorylated expression of ERK1/2 was analyzed by Western blotting. The mRNA expression of transforming growth factor β₁(TGF-β₁), bone morphogenetic protein 2(BMP-2) and core binding factor α1(Cbfα1) was measured by fluorescence quantitative PCR. RESULTS:QUE at concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L induced the expression of ALP in MSCs in a dose-dependent manner, and also promoted MSCs proliferation. The expression levels of ALP, BGP and ColⅠwere higher in QUE group, and was lower in PD89059 group than those in control group. Compared with control group, the level of phosphorylated ERK1/2, and the mRNA expression of TGF-β₁, BMP-2 and Cbfα1 increased in QUE group. The mRNA expression of TGF-β₁, BMP-2 and Cbfα1 in QUE+PD98059 group decreased as compared with QUE group. CONCLUSION:QUE promotes osteogenic differentiation of MSCs by activating ERK signaling pathway.     

Effects of rhBMP-2/collagen composite on the remodeling of rat interparietal suture after rapid expansion

AIM: The purpose of this study was to investigate the effects of rhBMP-2/collagen composite on bone regeneration during expansion of the interparietal suture in the rats. METHODS: 32 10-week old SD rats were divided into groups consisting of 8 rats each. They were comprised of normal control group, expansion control group and the treatment group, the two treatment groups were covered with atelo-typeⅠcollagen and rhBMP-2/collagen composite on the suture before subjected to the expansion force. The bone regeneration in the interparietal suture was estimated by histological method, the osteocalcin content was measured by radioimmonoassay and the calcium content was determined by atomic absorption spectrophotometer. RESULTS: The bone regeneration was more active in the suture after giving an expanding force than in the suture without any intervention. Even bone bridge was formed in the rhBMP-2/collagen composite group. Both the osteocalcin content and calcium content were much higher in the rhBMP-2/collagen composite group than in other three groups (P<0.01). CONCLUSION: The study shows that rhBMP-2/collagen composite promotes the bone regeneration in the expanded suture. It's suggested that rhBMP-2/collagen composite may be of therapeutic benefit in inhibiting relapse and shorting the retention period through acceleration of bone regeneration in the midpalatal suture.     

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.     

Effects of different HA/ZrO2 composites on adherence,proliferation and osteogenesis of cultured MSCs

AIM: To study the adherence, proliferation and osteogenesis effects of different composites on cultured mesenchymal stem cells (MSCs). METHODS: Zirconium oxide (ZrO₂) with monolayer hydroxyapatite (HA) composite and ZrO₂ with gradient HA composite were prepared using dry-laying method. The surface topography of the composites was observed. The rabbit MSCs were isolated and cultured on HA/ZrO₂ monolayer composite, HA/ZrO₂ gradient composite, pure HA or pure ZrO₂ slices. The adherence, proliferation and osteogenesis of the MSCs were assayed. The activity of alkaline phosphatase was detected. The mRNA expression of collagen I, osteocalcin and osteopontin was determined by RT-PCR. RESULTS: The discontinuous or continuous HA surface was observed in HA/ZrO₂ monolayer composite,while the surface of prepared HA/ZrO₂ gradient composite was fairly rough with porosity. The X-ray diffraction analysis shows that after megatemperature sintering, the ZrO₂ phase on the surface of the composite still remained, while the HA phase transformed to β-Ca₃(PO₄)₂(β-TCP), α-Ca₃(PO₄)₂(α-TCP) and CaZrO₃ phases. Cell culture showed that the HA/ZrO₂ gradient composite was in favour of cell adherence. The alkaline phosphatase activity in MSCs on pure HA slice was significantly increased compared compared with other groups.The mRNA expression of collagen I, osteocalcin and osteopontin in MSCs on HA/ZrO₂ composites and pure HA silice was higher than that in control group,especially the expression of collagen I. CONCLUSION: The HA/ZrO₂ garded composite promotes the proliferation of MSCs to a certain extent, and also promotes the osteogenesis differentiation of MSCs.     

Runx2 promotes osteogenic differentiating C2C12 cells through inhibiting macroautophagy

AIM：To explore the relationship between macroautophagy and Runx2-induced osteogenic differentiation of C2C12 cells.METHODS：The cells of C2C12/Runx2Dox subline were used in the study, which expresses Runx2 in response to doxycycline (Dox). The cells were treated with Dox (10 mg/L) for 0 d, 1 d, 3 d, and 6 d, and the expression levels of LC3b, Beclin-1, p62, and LAMP-2 were detected at each time point using real-time qPCR. The LC3-I/LC3-II ratio was measured by Western blotting. The cells were treated with different concentrations of 3-methyladenine (3-MA) or rapamycin (Rap) for 14 days in the presence of Dox, and the alkaline phosphatase (ALP) activity was measured. The cells were treated with 3-MA (5 mmol/L) or Rap (10 μmol/L) for 1 d, 3 d, and 6 d in the presence of Dox, and the expression levels of ALP and osteocalcin (OC) were detected.RESULTS：The expression levels of LC3b and Beclin-1 were dramatically down-regulated during Runx2-induced osteogenic differentiation of C2C12 cells, and the levels of p62 and LAMP-2 were not changed during this process. The conversion of LC3-I into LC3-II was inhibited. The ALP activity was increased in 3-MA (5 mmol/L)-treated cells, but decreased in Rap (10 μmol/L)-treated cells. The expression levels of ALP and OC were up-regulated by 3-MA treatment and were down-regulated by Rap treatment.CONCLUSION：Runx2 induces osteogenic differentiation of C2C12 cells by inhibiting the formation of autophagosome through down-regulating LC3 and Beclin-1 as well as inhibiting the conversion of LC3-I to LC3-II.     

平菇培养料发酵度快速测定指标的建立

为建立平菇培养料发酵度的判定指标,选用以粉碎玉米芯小粒、中粒和大粒为主要原料配制的平菇培养料,分析了发酵过程中含水量、pH值和总碳、总氮、水溶性有机碳(DOC)、氨含量以及杂菌抑制物质含量的变化,以及栽培平菇的生产特性.结果表明,发酵过程中,3种培养料的pH值先降后升,含水量、总碳含量、C/N和DOC含量降低,氨含量先...     

Activation of smad signaling and collagenⅠsynthesis in NRK52E cells induced by advanced glycation end products

AIM:To investigate the effects of advanced glycation end products on activation of Smad signaling pathway and collagenⅠ synthesis in proximal tubular epithelial cells. METHODS:Advanced glycation end products (AGE-BSA) were prepared by incubation of bovine serum albumin (BSA) with D-glucose. Normal rat proximal tubular epithelial (NRK52E) cells were cultured in RPMI-1640 medium with AGE-BSA. Phosphorylation and nuclear translocation of Smad2/3 were examined by immunocytochemistry. Levels of TGF-β1 in supernatant of cell culture were measured by enzyme-linked immunosorbent assay (ELISA). Expression of TGF-β1, Smad2, Smad3 and Smad7 mRNA were detected by RT-PCR. Expression of α-SMA , E-cadherin and collagenⅠproteins were detected by Western blotting.RESULTS:AGE-BSA induced Smad2/3 phosphorylation and nuclear translocation, two peaks occured at 30 min (68% vs 16%, P<0.05) and 24 h (76% vs 16%, P<0.05) compared to 0 min. The level of TGF-β1 markedly increased in supernatant of cell culture by induced AGE-BSA at 24 h and 48 h. The expression of TGF-β1 mRNA markedly increased at 24 h, and associated with high expression of Smad2, Smad3 and Smad7 mRNA at 48 h. AGE-BSA up-regulated significantly the expression of α-SMA and collagenⅠproteins, down-regulated the expression of E-cadherin protein. CONCLUSION:AGEs induces activation of Smad signaling, as well as transdifferentiation and collagenⅠ synthesis in proximal tubular epithelial cells.     

Role of angiotensin Ⅱ on typeⅠcollagen synthesis and its mRNA expression in vascular adventitial fibroblasts

AIM:Effects of angiotensin Ⅱ on typeⅠcollagen synthesis and its mRNA expression in cultured vascular adventitial fibroblasts. METHODS:Vascular adventitial fibroblasts (VAF)were isolated, cultured from rat thoracic aorta by explant method. ELISA was used to study typeⅠcollagen synthesis and competitive RT-PCR was employed to detect its mRNA expression after angiotensin Ⅱ administration. RESULTS: Angiontensin Ⅱ caused a dose dependent increase of typeⅠcollagen synthesis and its mRNA expression in VAF. CONCLUSION:The results support that angiotensin Ⅱ is an important factor controlling collagen metabolism of VAF and VAF may play an important role in vascular remodelling of hypertension.     

Effect of exogenous hydrogen sulfide on lipophagy in mouse primary hepatocytes

AIM: To evaluate the effect of exogenous hydrogen sulfide (H₂S) from GYY4137 on lipophagy in mouse primary hepatocytes. METHODS: The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups: the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h; the cells in H₂S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h. The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope, phase-contrast microscope or transmission electron microscope. The protein expression of LC3-Ⅰ/Ⅱ in the hepatocytes was determined by Western blot. RESULTS: In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H₂S group increased. CONCLUSION: In steatosis hepatocytes, exogenous H₂S promotes the lipophagy.     

《中国蔬菜品种志》

AIM: To characterize the gene expression of sortilin on adipogenic and osteogenic differentiation in mesenchymal stem cells (MSCs) in vitro and explore its significance.METHODS: MSCs derived from human bone marrow were isolated and cultured in vitro, then were stimulated in osteogenic medium and adipogenic medium, respectively. Osteopontin and lipoprotein lipase were detected by RT-PCR. Sortilin expression was analyzed by semiquantitative RT-PCR. RESULTS: 1.MSCs displayed the potential of differentiation into osteoblast and adipocyte. 2.Sortilin was upregulated one day after osteogenic induction and remained upregulated for a week. The expression of sortilin was significant increased on day 3(P＜0.01). 3. No significant changes of sortilin expression was found in adipogenic differentiation (P＞0.05).CONCLUSION: Sortilin may be useful to modulate the osteogenic differentiation and may not be necessary for adipocyte commitment in MSCs. The regulation of sortilin expression may provide new protocal and strategy for the treatment of osteoporosis and osteopenic disease.     

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.     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.     

Effects of diammonium glycyrrhizinate on the lipid peroxidation and interstitial collagenase activity in fibrotic liver in rats

AIM: To investigate the effect of diammonium gycyrrhizinate (Ganlixin) against liver fibrosis through preventing lipid peroxidation and regulating interstitial collagenase activity. METHODS: The liver fibrotic model was induced through subcutaneous injection of CCl4 and the feeding with high fat and low protein in rats. Diammonium gycyrrhizinate was administered (70 mg/kg rat BW). Hepatic inflammation and collagen were observed with H-E and Sirius red staining. The liver function including serum ALT, AST activity, Alb and total bilirubin levels were determined. The hepatic lipid peroxidation including SOD and GSH-Px activities, MDA and GSH content were also measured. Hepatic hydroxyproline (Hyp) content was detected with Jamall's method. The activity of interstitial collagenase in liver was assayed by the reaction with ［3H］ labeled type I collagen, and the gene expression of α1(Ⅰ) pro-collagen was analyzed by RT-PCR. RESULTS: The model rats had remarkable inflammatory necrosis, collagen accumulation and fibrosis at liver, while the diammonium gycyrrhizinate treated group showed slighter hepatic injury and collagen deposition, and the much better liver function than the model. The diammonium gycyrrhizinate-treated group had lower levels of hepatic MDA, Hyp and α1 (Ⅰ) pro-collagen mRNA expression than those in the model group, but had higher levels of interstitial collagenase activity, GSH content, SOD and GSH-Px activity than those in the model group. CONCLUSION: Diammonium gycyrrhizinate has a good effect against liver fibrosis, which may be related to the prevention from lipid peroxidation and improvement of interstitial collagenase activity in fibrotic livers.     

Effects of MEG3 and adenosine on autophagy and proliferation of HepG2 cells

AIM:To study the effects of maternal expressed gene 3 (MEG3) and adenosine on the autophagy and proliferation of human hepatocellular carcinoma HepG2 cells, and to explore the possible mechanisms of autophagy and the effect on the proliferation of HepG2 cells induced by MEG3 and adenosine. METHODS:HepG2 cells were cultured according to the conventional cultural method, and divided into control group, MEG3 group (the cells were transfected with MEG3 lentivirus), adenosine group (the cells were treated with 1 mmol/L adenosine) and MEG3+adenosine group (the cells were treated with 1 mmol/L adenosine and MEG3 lentivirus). The protein expression of LC3-Ⅱ, LC3-Ⅰ and mammalian target of rapamycin (mTOR) was determined by Western blot. MDC staining was used to observe the number of autophagosomes. The cell viability was measured by CCK-8 assay, and the number of viable cells were counted by automated cell counter. RESULTS:Compared with control group, LC3-Ⅱ expression and the ratio of LC3-Ⅱ/LC3-Ⅰ were decreased, mTOR expression was increased (P<0.05), the viability of HepG2 cells was decreased, and the number of autophagosomes were reduced in MEG3 group and adenosine group. In MEG3+adenosine group, LC3-Ⅱ expression and the ratio of LC3-Ⅱ/LC3-Ⅰ were decreased significantly (P<0.01), mTOR expression was increased significantly (P<0.01), and the viability and autophagosomes of HepG2 cells were reduced markedly as compared with MEG3 group and adenosine group. After treated with MEG3 and adenosine for 24~72 h, the viable HepG2 cells reduced significantly in MEG3 group and adenosine group (P<0.01), especially in MEG3+adenosine group (P<0.01). CONCLUSION:MEG3 overexpression and low concentration of adenosine activate the mTOR pathway, and inhibit the autophagy and proli-feration of HepG2 cells. MEG3 enhances the effect of adenosine on HepG2 cells.     

Knockdown of HMGA2 gene inhibits TGF-β1-induced human embryonic lung fibroblast growth and collagen synthesis

AIM: To investigate the effects of high mobility group A2(HMGA2) gene knockdown on the cell viability, apoptosis, collagen synthesis and oxidative stress of human embryonic lung fibroblast (HELF) induced by transforming growth factor-β1 (TGF-β1). METHODS: The HELF were divided into blank group, TGF-β1 group,negative control (NC) group and HMGA2 siRNA(si-HMGA2) group. The protein levels of HMGA2, AKT and p-AKT were determined by Western blot. The cell viability and apoptotic rate was analyzed by MTT assay and flow cytometry,respectively. The mRNA expression of collagen I (COL-Ⅰ) and COL-Ⅲ was detected by RT-qPCR. DCFH-DA was used to detect the content of reactive oxygen species (ROS). RESULTS: Compared with blank group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in TGF-β1 group were significantly increased, but the apoptotic rate and ROS level were significantly decreased (P<0.05). Compared with TGF-β1 group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in si-HMGA2 group were significantly decreased, but the apoptotic rate and ROS level were significantly increased (P<0.05). CONCLUSION: Knockdown of HMGA2 gene expression decreases the viability and collagen synthesis, and promotes apoptosis and ROS production of human embryonic lung fibroblasts induced by TGF-β1. The mechanism may be related to down-regulation of PI3K/AKT signaling pathway.     

Effects of endogenous carbon monoxide on the expression of transforming growth factor-beta 3 and type Ⅰ collagen of pulmonary artery in rats under hypoxia

AIM: To explore the impact of endogenous carbon monoxide (CO) on the expression of transforming growth factor-beta 3 (TGF-β₃) and type Ⅰcollagen in pulmonary artery of rats under hypoxia. METHODS: In the model of rats under hypoxic pulmonary hypertension, the measurement of pulmonary artery mean pressure (PAMP) and carboxyhemoglobin (HbCO) formation within pulmonary tissue homogenates was performed. TGF-β₃ and collagen Ⅰexpressions were detected by immunohistochemical assay. The expressions of TGF-β₃, type Ⅰ procollagen mRNA, and tissue inhibitor of metalloproteinase -1 (TIMP-1) mRNA were detected by in situ hybridization. RESULTS: ZnPP significantly increased PAMP and markedly decreased HbCO formation within lung tissue homogenates in rats under hypoxia( P< 0.01). Meanwhile, ZnPP promoted the expression of TGF-β₃ and collagen Ⅰprotein in pulmonary arteries in rats under hypoxia ( P< 0.01). ZnPP obviously elevated the expressions of TGF-β₃ mRNA, type Ⅰ procollagen mRNA, and TIMP-1 mRNA in pulmonary arteries in rats under hypoxia ( P< 0.01). CONCLUSION: Endogenous CO plays an important role in decreasing collagen synthesis and promoting degradation in pulmonary artery of rats under hypoxia by inhibiting the expression of TGF-β₃.     

Role of TGF-β1 and its signaling transduction molecule Smad4 in the development of glomerulosclerosis

AIM: To explore the role of TGF-β1 and signaling transduction molecule， Smad4， in the development of glomerulosclerosis. METHODS: Expression levels of TGF-β1, Smad4, collagen Ⅰ proteins were evaluated by immunohistochemistry in renal biopsies from 38 cases with a spectrum of glomerulonephritis, and compared with 20 normal kidney tissue with image analysis system. After stimulation with TGF-β1, expressions of endogenous Smad4 and collagen Ⅰ mRNA and proteins and its modulation by TGFβ1 were evaluated by RT-PCR and Western blotting analyses in cultured human mesangial cells. RESULTS: All types of proliferative and sclerotized glomerulonephritis showed an increased expression of TGF-β1, Smad4 and collagen Ⅰ ompared with the 20 normal kidney tissue in glomerular (P<0.05). The expression of TGF-β1 and Smad4 proteins were correlation with deposition of collagen Ⅰ (P<0.05). The expression of Smad4 mRNA and protein were elevated by TGF-β1 in a dose and time-dependent manner (P<0.05), and the expression of collagen Ⅰ mRNA and protein were also increased both in a dose and time-dependent manner (P<0.05). CONCLUSION: Our data indicate that TGF-β1 and its signaling transduction molecule (Smad4 proteins) may be involved in the excessive deposition of extracellular matrix in glomerular disease and play an important role in the genesis and progress of glomerulosclerosis.