Role of PI3K/Akt/mTOR pathway-mediated macrophage autophagy in affecting the phenotype transformation of lung fibroblasts induced by silica dust exposure / 中南大学学报(医学版)

OBJECTIVES@#The phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway is one of the main signaling pathways related to autophagy. Autophagy plays a key role in the formation of silicosis fibrosis. The phenotypic transformation of lung fibroblasts into myofibroblasts is a hallmark of the transition from the inflammatory phase to the fibrotic phase in silicosis. This study aims to investigate whether the PI3K/Akt/mTOR pathway affects the phenotypic transformation of silicosis-induced lung fibroblasts into myofibroblasts via mediating macrophage autophagy.@*METHODS@#The human monocytic leukemia cell line THP-1 cells were differentiated into macrophages by treating with 100 ng/mL of phorbol ester for 24 h. Macrophages were exposed to different concentrations (0, 25, 50, 100, 200, 400 μg/mL) and different times (0, 6, 12, 24, 48 h) of SiO2 dust suspension. The survival rate of macrophages was measured by cell counting kit-8 (CCK-8) method. Enzyme linked immunosorbent assay (ELISA) was used to measure the contents of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) in the cell supernatant. The co-culture system of macrophages and HFL-1 cells was established by transwell. A blank control group, a SiO2 group, a LY294002 group, a SC79 group, a LY294002+SiO2 group, and a SC79+SiO2 group were set up in this experiment. Macrophages in the LY294002+SiO2 group were pretreated with LY294002 (PI3K inhibitor) for 18 hours, and macrophages in the SC79+SiO2 group were pretreated with SC79 (Akt activator) for 24 hours, and then exposed to SiO2 (100 μg/mL) dust suspension for 12 hours. The expression of microtubule-associated protein 1 light chain 3 (LC3) protein in macrophages was detected by the immunofluorescence method. The protein expressions of PI3K, Akt, mTOR, Beclin-1, LC3 in macrophages, and collagen III (Col III), α-smooth muscle actin (α-SMA), fibronectin (FN), matrix metalloproteinase-1 (MMP-1), tissue metalloproteinase inhibitor-1 (TIMP-1) in HFL-1 cells were measured by Western blotting.@*RESULTS@#After the macrophages were exposed to SiO2 dust suspension of different concentrations for 12 h, the survival rates of macrophages were gradually decreased with the increase of SiO2 concentration. Compared with the 0 μg/mL group, the survival rates of macrophages in the 100, 200, and 400 μg/mL groups were significantly decreased, and the concentrations of TGF-β1 and TNF-α in the cell supernatant were obviously increased (all P<0.05). When 100 μg/mL SiO2 dust suspension was applied to macrophages, the survival rates of macrophages were decreased with the prolonged exposure time. Compared with the 0 h group, the survival rates of macrophages were significantly decreased (all P<0.05), the concentrations of TGF-β1 and TNF-α in the cell supernatant were significantly increased, and the protein expression levels of Beclin-1 and LC3II were increased markedly in the 6, 12, 24, and 48 h groups (all P<0.05). Immunofluorescence results demonstrated that after exposure to SiO2 (100 μg/mL) dust for 12 h, LC3 exhibited punctate aggregation and significantly higher fluorescence intensity compared to the blank control group (P<0.05). Compared with the blank control group, the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated in the SiO2 group (all P<0.05). Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were down-regulated and the protein expressions of LC3II and Beclin-1 were up-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were decreased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were down-regulated (all P<0.05) in the LY294002+SiO2 group. Compared with the SiO2 group, the protein expressions of PI3K, Akt, and mTOR were up-regulated and the protein expressions of LC3II and Beclin-1 were down-regulated in macrophages (all P<0.05), the contents of TNF-α and TGF-β1 in the cell supernatant were increased (both P<0.01), and the protein expressions of Col III, FN, α-SMA, MMP-1, and TIMP-1 in HFL-1 cells were up-regulated (all P<0.05) in the SC79+SiO2 group.@*CONCLUSIONS@#Silica dust exposure inhibits the PI3K/Akt/mTOR pathway, increases autophagy and concentration of inflammatory factors in macrophages, and promotes the phenotype transformation of HFL-1 cells into myofibroblasts. The regulation of the PI3K/Akt/mTOR pathway can affect the autophagy induction and the concentration of inflammatory factors of macrophages by silica dust exposure, and then affect the phenotype transformation of HFL-1 cells into myofibroblasts induced by silica dust exposure.

Toxicity and Efficacy of Water-eluted Fraction from Siegesbeckiae Herba Against Human Embryonic Lung Fibroblasts / 中国实验方剂学杂志

Objective:To detect the toxicity of water-eluted fraction from Siegesbeckiae Herba （SWEF） at different concentrations against MRC-5 human embryonic lung fibroblasts and its impacts on the expression of <italic>α</italic>7 nicotinic acetylcholine receptor （<italic>α</italic>7nAChR） and inflammatory factors， so as to figure out the active components responsible for toxicity and efficacy. Method:The toxicities of SWEF at 1， 6， 10， 20， and 50 g·L^-1 against MRC-5 cells were determined by cell counting kit-8 （CCK-8） assay combined with flow cytometry and Trypan blue staining. The changes in <italic>α</italic>7nAChR expression and inflammatory factor levels before and after <italic>α</italic>7nAChR gene silencing were detected to reveal the pharmacodynamic effect of SWEF on MRC-5 cells. Result:SWEF （≥6 g·L^-1） obviously inhibited the viability of MRC-5 cells （<italic>P</italic><0.01） and promoted their apoptosis and necrosis （<italic>P</italic><0.01）， with the half-maximal inhibitory concentration （IC₅₀） being 6.03 g·L^-1. The determination of <italic>α</italic>7nAChR expression and inflammatory factor levels in MRC-5 cells showed that SWEF contained <italic>α</italic>7nAChR agonist-like substance， which enhanced <italic>α</italic>7nAChR mRNA and protein expression （<italic>P</italic><0.05， <italic>P</italic><0.01） and decreased the inflammatory factor levels （<italic>P</italic><0.05， <italic>P</italic><0.01）. SWEF down-regulated the inflammatory factors possibly by re-regulating <italic>α</italic>7nAChR mRNA expression， exhibiting a negative correlation between them （<italic>P</italic><0.01）. Conclusion:SWEF （≥6 g·L^-1） is highly toxic to MRC-5 cells. Pharmacodynamic studies have confirmed that <italic>α</italic>7nAChR agonist-like substance contained in SWEF was responsible for the elevated <italic>α</italic>7nAChR expression and reduced inflammatory cytokines. It is inferred that excessive <italic>α</italic>7nAChR agonist-like substance may trigger the toxicity of<italic> </italic>Siegesbeckiae Herba.

Effect of Jianpi Xiaoai Prescription on Activation of HFL1 into CAFs Induced by Exosomes in Colon Cancer / 中国实验方剂学杂志

Objective:To observe the effect of Jianpi Xiaoai prescription on the activation of normal human embryonic lung fibroblasts （HFL1） into tumor-associated fibroblasts （CAFs） induced by human colon cancer cells （HCT116） derived exosomes. Method:SD rats were gavaged with 13.1 g·kg-1 of Jianpi Xiaoai prescription to prepare drug-containing serum， and HCT116 cell exosomes-containing 10% exosomes-free serum and 20% Jianpi Xiaoai prescription drug serum were isolated by ultra-high speed centrifugation. The particle size distribution of exosomes were detected by Nanoparticle tracking analyzer （Zetaview）， and the exosomes' marker proteins apoptotic transfer gene 2 interaction protein X （Alix）， heat shock protein 70 （HSP70）， and tumor-susceptibility gene 101 （TSG101） were identified by Western blot， and the uptake of exosomes labeled with cell membrane staining kit （PKH67） by HFL1 was observed by fluorescence microscope. HFL1 cells were divided into six groups： the blank group， the transforming growth factor-β1 （TGF-β1） group， the TGF-β1 combined with HCT116 exosomes of 2 mg·L-1 group， the TGF-β1 combined with HCT116 exosomes of 4 mg·L-1 group， the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 2 mg·L-1 group， and the TGF-β1 combined with Jianpi Xiaoai prescription exosomes of 4 mg·L-1 group， and all groups were cultivated for 48 h. Western blot and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） were used to determine the protein and mRNA expressions of α-smooth muscle actin （α-SMA）. Result:The particle size distribution detected by Zetaview was mainly between 50-100 nm， and the exosomes were verified based on the expressions of marker proteins Alix， HSP70 and TSG101. After co-incubation of HFL1 cells with exosomes， a large number of exosomes were absorbed by HFL1 cells under fluorescence microscope. Compared with the blank control group， the protein and mRNA expressions of α-SMA in the TGF-β1 group and TGF-β1 combined with HCT116 exosome groups were increased （P<0.01）. Compared with the TGF-β1 combined with HCT116 exosome groups， the protein and mRNA expressions of α-SMA were decreased in the TGF-β1 combined with Jianpi Xiaoai prescription exosome groups （P<0.01）. Conclusion:Human colon cancer cell exosomes combined with TGF-β1 can induce the activation of HFL1 into CAFs， and Jianpi Xiaoai prescription can reduce the activation of HFL1 by affecting the expressions of α-SMA， thus antagonizing the lung metastasis of colon cancer.

A study on alterations in mitochondrial biological characteristics during cellular senescence of human embryonic lung fibroblasts / 中华预防医学杂志

Objective@#To study the alterations of mitochondrial biological characteristics during both cellular replicative and premature senescence induced by hydrogen peroxide in human embryonic lung fibroblasts (HEFs).@*Methods@#The premature senescence was induced by 400 μmol/L H2O2 once a day at the same time and with 2 hours each time, after four consecutive days the premature senescence models were classified into premature senescence initiation group (PSi) and premature senescence persistence group (PSp). Based on the life span of HEFs, the cell replicative senescence was divided into five groups included young-age (22 PDL), middle-age (35 PDL), replicative senescence (49 PDL), PSi and PSp. The mitochondrial distribution, relative content, adenosine triphosphate (ATP) contents, 8-hydroxydeoxyguanosine (8-OHdG) levels, the relative mitochondrial transcription factor A (TFAM) as well as mitochondrial DNA methyltransferase 1 (mtDNMT1) mRNA levels, mtDNA copy number, the relative TFAM protein level and the total enzyme activity of mitochondrial DNA methyltransferases (mtDNMTs) were detected in five senescence groups.@*Results@#The mtDNA copy number, 8-OHdG contents, level of mtDNMT1 mRNA and mtDNMTs activity in 49 PDL group were higher than those in 22 PDL group (all P values <0.05); The level of 8-OHdG in PSi was higher than that in 22 PDL group (P<0.05); The ATP contents, mtDNA copy number, the mRNA and protein expression levels of TFAM and mtDNMTs activity of PSp were higher than those in 22 PDL group (all P values<0.05).@*Conclusion@#During the cellular senescence of HEFs, the higher mtDNA copy number and mtDNMTs activity were common features regardless of replicative or premature senescence, with possibility that oxidative stress was involved in modifying the occurrence of premature senescence.

Effects of Rapamycin and mammalian target of rapamycin - small interfering RNA (mTOR siRNA)on proli-feration,apoptosis and extracellular matrix in premature rats lung fibroblasts exposed to hyperoxia / 中华实用儿科临床杂志

Objective To investigate the effects of Rapamycin and mammalian target of rapamycin - small in-terfering RNA (mTOR siRNA)on the proliferation,apoptosis and collagen Ⅰ(COLⅠ),collagen Ⅲ(COLⅢ)and fi-bronectin(FN)in premature rats lung fibroblasts exposed to hyperoxia. Methods 900 mL/ L volume fraction of oxygen was used to establish hyperoxia - damaged cell models,and the premature rats lung fibroblasts were divided into air control group,hyperoxia group,hyperoxia + rapamycin group and mammalian target of rapamycin - small interfering RNA transfection group. Cell proliferation was assessed by using 3 -(4,5 - Dimethylthiazol - 2 - yl)- 2,5 - dipheny-ltetrazolium bromide assay. Apoptosis were detected by Annexin V - FITC and propidium lodide (PI)double staining. The expressions of COLⅠ,COLⅢ and fibronectin was assessed by using enzyme linked immunosorbent assay and Bcl - 2,P53 and pro - fibrotic factors of connective tissue growth factor(CTGF)and transforming growth factor β(TGF - β)by using Western blot. Results Compared with the air control group,the proliferation of lung fibroblasts decreased and the apoptosis increased in the hyperoxia group,while the contents of COLⅠ(28. 30 ± 0. 53 vs. 17. 43 ±0. 37),COLⅢ(27. 86 ± 1. 02 vs. 17. 43 ± 0. 37)and fibronectin(32. 87 ± 0. 42 vs. 21. 57 ± 0. 47),P53(0. 810 ± 0. 119 vs. 0. 160 ± 0. 018),TGF - β(0. 580 ± 0. 108 vs. 0. 210 ± 0. 008)and CTGF(0. 590 ± 0. 017 vs. 0. 220 ± 0. 007)were also increased but the expression of Bcl - 2(0. 150 ± 0. 004 vs. 0. 600 ± 0. 130)protein was decreased, and the differences were all statistically significant (all P < 0. 01). Compared with the hyperoxia group,the proliferation of lung fibroblasts was increased in the hyperoxia + rapamycin group,but the apoptosis was decreased,the contents of COLⅠ(23. 17 ± 0. 60 vs. 28. 30 ± 0. 53),COLⅢ(17. 09 ± 0. 58 vs. 27. 86 ± 1. 02)and fibronectin(28. 11 ± 0. 68 vs. 32. 87 ± 0. 42),P53(0. 430 ± 0. 008 vs. 0. 810 ± 0. 119),TGF - β(0. 380 ± 0. 008 vs. 0. 580 ± 0. 108)and CTGF (0. 040 ± 0. 006 vs. 0. 590 ± 0. 017)were decreased while the expression of Bcl - 2(0. 290 ± 0. 009 vs. 0. 150 ± 0. 004) protein was increased,and the differences were all statistically significant (all P < 0. 01). In the mTOR siRNA transfec-tion group,compared with the hyperoxia + rapamycin group,the proliferation of lung fibroblasts was increased,but the apoptosis was decreased;the contents of COLⅠ(15. 71 ± 0. 34 vs. 23. 17 ± 0. 60),COLⅢ (13. 85 ± 1. 36 vs. 17. 09 ± 0. 58)and fibronectin(20. 18 ± 0. 28 vs. 28. 11 ± 0. 68),P53(0. 300 ± 0. 006 vs. 0. 430 ± 0. 008),TGF - β(0. 150 ± 0. 002 vs. 0. 380 ± 0. 008)and CTGF(0. 140 ± 0. 004 vs. 0. 040 ± 0. 006)were decreased while the expression of Bcl - 2 (0. 460 ± 0. 012 vs. 10. 290 ± 0. 009)protein was increased,and the differences were all statistically significant (all P < 0. 01). Conclusion Rapamycin and mTOR siRNA can protect lung injury caused by hyperoxia and have a certain inhibitory effect on pulmonary fibrosis,and mTOR siRNA effect is more obvious,so the mechanism may be through the inhibition of mTOR signaling pathway.

Effect of CKLF1-C19 polypeptide on differentiation of human lung fibro-blast into myofibroblast induced by TGF-β / 中国病理生理杂志

AIM:To investigate the effect of CKLF1-C19 polypeptide (C19) on differentiation of human lung fibroblast (LFB) into myofibroblast (MFB) induced by TGF-β. METHODS:LFBs were cultured and identified. LFBs were treated with TGF-β(5 μg/L) to establish the cell model of LFB differentiate into MFB. The LFBs were divided into 6 experimental groups including control group,TGF-β group,and TGF-β plus different doses(1,0.1,0.01,0.001 mg/L) C19 groups. The cell morphology,cell proliferation rate, and the expression of α smooth muscle actin (α-SMA) and collagen Ⅰ were observed. RESULTS:Human primary LFB was successfully cultured and was confirmed by the method of immunofluorescence. TGF-β at 5 μg/L induced proliferation and differentiation of LFB. The mRNA levels of α-SMA and collagen Ⅰ in TGF-β group were higher than that in control group(P<0.05).The cell proliferation rates,mRNA levels of α-SMA and collagen Ⅰ, and the protein expression of α-SMA in 0.01 mg/L+TGF-β group and 0.001 mg/L+TGF-β group were markedly lower than those in TGF-β group(P<0.05). CONCLUSION:C19 at 0.01 mg/L and 0.001 mg/L effectively inhibits differentiation of LFB into MFB induced by TGF-β, thus inhibiting the process of airway remodeling and fibrosis to some extent.

Effect of Dexamethasone on proliferation and expression of fibroblast growth factor mRNA in human embryo lung fibroblasts exposed to hyperoxia / 中华实用儿科临床杂志

Objective To explore the effect of Dexamethasone(DEX)on the proliferation and the expression of fibroblast growth factor(FGF)- 1,FGF - 2 and interleukin - 6(IL - 6)mRNA in hyperoxia - exposed human em-bryo lung fibroblasts(MRC - 5). Methods High oxygen volume fraction of 950 mL/ L was used to establish hyperoxia -damaged cell models,then treated with different concentrations of DEX(10 - 4 ,10 - 6 ,10 - 8 ,10 - 9 ,10 - 10 ,10 - 11 ,10 - 12 mol/ L),respectively. The proliferation activity of the cells was evaluated by methyl thiazolyl tetrazolium(MTT)at 24 h,48 h,72 h,and the optimal hyperoxia - exposed time and concentration of DEX were chosen;then they were divided into air group,hyperoxia group and hyperoxia + DEX group. The mRNA expressions of FGF - 1,FGF - 2 and IL - 6 were detected by quantitative real - time PCR at 48 h. Results Cell proliferation activity of the hyperoxia group was lower than that of the air group and there were significant differences at 48 h and 72 h(all P ﹤ 0. 05). Compared with the hyperoxia group,cell proliferation activity of the hyperoxia + DEX group increased with the concentration of DEX decreasing,reaching the peak at 10 - 9 mol/ L,and then gradually decreased with the concentration of DEX decreasing. Cells were cultured for 48 h,compared with the air group,the level of FGF - 1 mRNA was lower,FGF - 2 and IL - 6 mRNA were higher in the hyperoxia group(P ﹤ 0. 05). Compared with hyperoxia group,the level of FGF - 1 mRNA was higher,FGF - 2 and IL - 6 mRNA were lower in hyperoxia + DEX group(P ﹤ 0. 05). Conclusions Exposure to high oxygen volume fraction of 950 mL/ L for 48 h can cultivate optimal hyperoxia - damaged cell models,and DEX can protect the cell from hyperoxia injury and 10 - 9 mol/ L was the optimal concentration. Enhanced the expression of FGF - 1 mRNA and inhibited expression of FGF - 2 mRNA may be one of the mechanism that DEX protects the cells from hyperoxia injury.

Role of insulin-like growth factor II receptor in transdifferentiation of free silica-induced primary rat lung fibroblasts / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the role of insulin-like growth factor II receptor in free silica-induced transdifferentiation of primary rat lung fibroblasts.</p><p><b>METHODS</b>Rat lung fibroblasts and rat alveolar macrophages were cultured. A transdifferentiation model of primary rat lung fibroblasts was induced by free silica. Levels of α-SMA protein, IGF-IIR protein and mRNA were measured by immunocytochemistry, Western blot and RT-PCR, respectively. Lung fibroblasts were treated with Wortmannin.</p><p><b>RESULTS</b>The expression levels of α-SMA and IGF-IIR increased with the increasing free silica concentration and decreased after Wortmannin was used.</p><p><b>CONCLUSION</b>The IGF-IIR plays an important role in free silica-induced transdifferentiation of primary rat lung fibroblasts.</p>

Effects of paraquat on proliferation and viscoelasticity of human embryonic lung fibroblasts / 医用生物力学

Objective To study effects of paraquat on proliferation and viscoelasticity of human embryonic lung fibroblasts (MRC-5), and to discuss mechanism of MRC-5 damage at initial stage and pulmonary fibrosis later after paraquat intoxication. Methods MRC-5 cells were treated by culture medium with different concentration of paraquat (50, 100, 200 mg/L, respectively) for 12 hours, when the paraquat culture medium was replaced by normal culture medium. At 48th hours, MRC-5 cells were collected, examined and analyzed by flow cytometry for indicating the proliferation, and micropipette aspiration technique was used to investigate viscoelasticity of the cells. Results After treated by paraquat with different concentration, proliferation index (PI) of MRC-5 cells were significantly reduced, as compared with the control group (P0.05). Conclusions MRC-5 cells were damaged at initial stage of paraquat intoxication, with PI and viscoelastic parameters reducing. Decompensated repair after paraquat intoxication is an important reason leading to pulmonary fibrosis, which provides a new thought in clinical treatment.

Rapamycin Inhibits Transforming Growth Factor beta1-Induced Fibrogenesis in Primary Human Lung Fibroblasts

PURPOSE: The present study was designed to determine whether rapamycin could inhibit transforming growth factor beta1 (TGF-beta1)-induced fibrogenesis in primary lung fibroblasts, and whether the effect of inhibition would occur through the mammalian target of rapamycin (mTOR) and its downstream p70S6K pathway. MATERIALS AND METHODS: Primary normal human lung fibroblasts were obtained from histological normal lung tissue of 3 patients with primary spontaneous pneumothorax. Growth arrested, synchronized fibroblasts were treated with TGF-beta1 (10 ng/mL) and different concentrations of rapamycin (0.01, 0.1, 1, 10 ng/mL) for 24 h. We assessed m-TOR, p-mTOR, S6K1, p-S6K1 by Western blot analysis, detected type III collagen and fibronectin secreting by ELISA assay, and determined type III collagen and fibronectin mRNA levels by real-time PCR assay. RESULTS: Rapamycin significantly reduced TGF-beta1-induced type III collagen and fibronectin levels, as well as type III collagen and fibronectin mRNA levels. Furthermore, we also found that TGF-beta1-induced mTOR and p70S6K phosphorylation were significantly down-regulated by rapamycin. The mTOR/p70S6K pathway was activated through the TGF-beta1-mediated fibrogenic response in primary human lung fibroblasts. CONCLUSION: These results indicate that rapamycin effectively suppresses TGF-beta1-induced type III collagen and fibronectin levels in primary human lung fibroblasts partly through the mTOR/p70S6K pathway. Rapamycin has a potential value in the treatment of pulmonary fibrosis.

Retinoic Aacid Diminished the Expression of MMP-2 in Hyperoxia-exposed Premature Rat Lung Fibroblasts through Regulating Mitogen-activated Protein Kinases / 华中科技大学学报（医学）（英德文版）

This study examined the effects of retinoic acid (RA),PD98059,SP600125 and SB203580 on the hyperoxia-induced expression and regulation of matrixmetalloproteinase-2 (MMP-2) and metalloproteinase-2 (TIMP-2) in premature rat lung fibroblasts (LFs).LFs were exposed to hyperoxia or room air for 12 h in the presence of RA and the kinase inhibitors PD98059 (ERK1/2),SP600125 (JNK1/2) and SB203580 (p38) respectively.The expression levels of MMP-2 and TIMP-2 mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).MMP-2 activity was measured by zymography.The amount of p-ERK1/2,REK1/2,p-JNK1/2,JNK1/2,p-p38 and p38 was determined by Western blotting.The results showed that:(1) PD98059,SP600125 and SB203580 significantly inhibited p-ERK1/2,p-JNK1/2 and p-p38 respectively in LFs; (2) The expression of MMP-2 mRNA in LFs exposed to hyperoxia was decreased after treatment with RA,SP600125 and SB203580 respectively (P＜0.01 or 0.05),but did not change after treatment with PD98059 (P＞0.05).Meanwhile,RA,PD98059,SP600125 and SB203580 had no effect on the expression of TIMP-2 mRNA in LFs exposed to room air or hyperoxia (P＞0.05); (3) The expression of pro- and active MMP-2 experienced no change after treatment with RA or SP600125 in LFs exposed to room air (P＞0.05),but decreased remarkably after hyperoxia (P＜0.01 or 0.05).SB203580 inhibited the expression of pro- and active MMP-2 either in room air or under hyperoxia (P＜0.01).PD98059 exerted no effect on the expression of pro- and active MMP-2 (P＜0.05).It was suggested that RA had a protective effect on hyperoxia-induced lung injury by down-regulating the expression of MMP-2 through decreasing the JNK and p38 activation in hyperoxia.

The impact of p53 gene on the apoptosis induced by NaAsO2 in human embryonic lung fibroblasts / 中国地方病学杂志

Objective To investigate the p53,Bax,bcl-2 gene in NaAsO2-induced human embryonic lung fibroblasts(HELF)apoptosis.Methods HELF was divided into HELF cells transfected with p53 plasmid(p53 group),HELF cells transfected with PC plasmid(PC group)and normal cultured HELF cells(normal group).The mRNA expression of p53,Bax and bcl-2 gene was detected by real-time PCR,the protein expression of p53,Bax and bcl-2 was assessed by immunohistochemical SABC and the cell apoptosis of HELF was detected by flow cytometry(FCM),in a 6-well plate and cultured for 48 hours,which was exposed to different doses(0,3,9,15mmol/L)NaAsO2 for 24 hours.Results The p53 gene mRNA expression level of p53 group(0.51±0.29)was lower than that of the normal group and PC group [ (1.00 ± 0.20), (1.32 ± 0.26), all P < 0.05 ]. The p53 protein expression level of p53 group(4.10 ± 1.20) was lower than the PC group and normal group[ (8.00 ± 1.63), (7.90 ± 1.79), allP < 0.05]. In p53 group, PC group, normal group exposed to 0,3,9,15 mmol/L NaAsO2 doses, the apoptotic rate [(0.57 ± 0.28)%, (22.91 ± 4.86)%, (40.05 ± 3.93)%, (44.87 ± 3.58)%; (0.65 ± 0.24)%, (14.09 ± 3.49)%,(20.31 ± 3.66)%, (32.42 ± 3.63)%; (0.56 ± 0.25)%, (12.14 ± 3.70)%, (19.61 ± 3.63)%, (30.43 ± 2.83)%], Bax mRNA expression level[(12.73 ± 3.96), (25.12 ± 6.42), (104.96 ± 26.77), (154.04 ± 30.52); (14.63 ± 3.57),(36.75 ± 3.67), (272.26 ± 66.11), (846.12 ± 243.36); (14.75 ± 5.65), (37.22 ± 11.27), (278.51 ± 37.42),(861.67 ± 369.29) ], Bax protein expression level [ ( 15.07 ± 0.83 ) %, ( 23.79 ± 3.99 ) %, (38.51 ± 1.58 ) %, (53.86 ±1.74)%;(15.43 ± 1.45)%,(36.11 ± 1.37)%, (56.86 ± 1.97)%, (76.09 ± 2.01)%; (15.20 ± 1.03)%,(35.25 ±1.09)%, (55.56 ± 2.17)%, (74.48 ± 2.85)% ] was respectively increased in a dose-dependent manner with the increased concentration of NaAsO2(all P < 0.05). The bel-2 mRNA expression level [ (443.00 ± 244.47), (156.79 ±53.18), (62.13 ± 13.66), (23.10 ± 6.44); (420.55 ± 110.77), (48.15 ± 10.02), (14.91 ± 6.53), (7.54 ± 2.62);(577.75 ± 123.22), (49.68 ± 10.11), (12.41 ± 1.28), (7.22 ± 1.89)], bcl-2 protein expression level[(47.20 ±3.77)%, (41.80 ± 2.94)%, (36.00 ± 2.36)%, (29.00 ± 2.91)%; (45.90 ± 4.15)%, (35.70 ± 2.77)%, (29.80 ±2.78)%, (24.80 ± 2.66)% ; (46.70 ± 3.47)%, (36.20 ± 2.90)%, (30.10 ± 3.21)%, (25.10 ± 2.28)% ] wasdecreased in a dose-dependent manner with the increased concentration of NaAsO2(all P < 0.05 ). In 3,9,15 mmol/L NaAsO2, apoptotic rate of p53 group, mRNA expression of bcl-2, protein expression of bcl-2 was higher than that ofnormal group and PC group, respectively (all P < 0.05), but mRNA expression of Bax, protein expression of Bax was respeetivelylower than that normal group and the PC group(P < 0.05 ). Conclusion p53 gene reduced the apoptosis induced by NaAsO2 in HELF, possibly by changing the apoptosis pathway.

Serum IL-22 level in asthmatic patients and expression of IL-22R1 in human airway cells / 第二军医大学学报

Objective: To examine the serum level of IL-22 in asthmatic patients and the expression of IL-22R1 in human airway epithelial cells, human airway smooth muscle cells, and lung fibroblasts, so as to explore the target cells of IL-22. Methods: The serum levels of IL-22 and IL-17 in 36 asthmatic patients and 20 normal control subjects were measured by enzyme-linked immunosorbent assay (ELISA). And lung function of the asthmatic patients was assessed by Gaeger spirometry. According to the values of FEV1/FVC and FEV1%, the patients were divided into two groups, bronchodilation test positive group (19 patients) and bronchial provocation test positive group (17 patients). The expression of IL-22R1 mRNA in human airway epithelial cells, human airway smooth muscle cells, and lung fibroblasts was examined using real-time PCR, and IL-22R1 protein expression was detected by immunofluorescence staining and Western blotting analysis. Results: There was no significant difference in serum IL-22 and IL-17 levels between the asthmatic patients and normal controls. The serum IL-22 and IL-17 levels in asthmatic patients positive for bronchodilation test was significantly higher than those positive for bronchial provocation test(P<0.05). IL-22R1 mRNA and protein were detected in all the 3 types of cells. Conclusion: IL-22 may be involved in the pathogenesis of asthma, and human airway epithelial cells, human airway smooth muscle cells, and lung fibroblasts may all be the target cells of IL-22.

The effects of acetylcysteine on proliferation and collagen synthesis of human lung fibroblast / 中国综合临床

Objective To study the effects of N-acetylcysteine (NAC) on the proliferation, apoptosis and collagen synthesis of human lung fibroblast (HLF). Methods HLF was primarily cultured in complete medium of DMEM/F12. Different concentrations of NAC (5,10,20,40mmol/L) were administrated. Cell proliferations were tested by methylthiazolyltetrazolium (MTT) ,apoptosis and cell cycle were detected with Flow cytometer and mRNA and 40 mmoi/L )after 24 hours,the apoptosis rates were (34.38±5.80)%, (37.72±3.10)%, (44.05± 4.52) % and (59.18±5.24) % ) respectively, significantly higher than that of the controls (3.92±1.24) % pression of type Ⅰ procollagen in HLF was decreased significantly after administration of NAC at 5, 10,20, 40 mmol/L respectively (P < 0.01 ). Conclusion Administration of NAC induces apoptosis and directly inhibites the proliferation and the collagen synthesis of HLF.

Effects of radical scavenger protein from broad beans on glutathione status in human lung fibroblasts

<p><b>OBJECTIVE</b>Human diploid cells are more susceptible to oxidative stress at late passage than at early passage, presumably because of the decrease in cellular-reduced glutathione (GSH) concentration. Water-soluble protein (WSP) from broad beans scavenges free radicals. The effects of WSP on the glutathione system were examined in PDL 20 (early passage) and PDL 50 (late passage) human lung fibroblasts (TIG-1).</p><p><b>METHODS</b>To determine cytosolic glutathione peroxidase (GSH-Px) activities, glutathione reductase (GR) activities, oxidized glutathione (GSSG) concentrations, and GSSG/reduced glutathione (GSH) ratios, WSP and hydrocortisone (HC) treatments of TIG-1 cells (PDL 20→50 and PDL 50→75) were performed for 40 days. We also investigated the GSSG concentrations and GR activities in PDL 20 cells that were continuously treated with WSP until PDL 39 and 55.</p><p><b>RESULTS</b>GSSG concentrations decreased in WSP- and HC-treated PDL 50→75 cells. The GSSG/GSH ratios in PDL 50→75 cells became low after the treatments. Increases in GR activities were observed in treated PDL 50→75 cells. The decline in the GSSG concentration of PDL 50→75 cells correlated with the increase in GR activity. The GSSG levels in control cells were higher following cellular age, whereas the levels in treated cells were lower than those in the control. The studies on cellular age-related changes indicated that greater increases in GR activity were found in treated cells than in the control.</p><p><b>CONCLUSION</b>These results indicated that WSP influences the GSSG concentration that is associated with cellular aging, but the mechanism of GSSG reduction by WSP remains unknown. The enhancement of glutathione status following WSP treatment may be related to the delay in the cellular aging.</p>

Effects of Radical Scavenger Protein from Broad Beans on Glutathione Status in Human Lung Fibroblasts

Objective: Human diploid cells are more susceptible to oxidative stress at late passage than at early passage, presumably because of the decrease in cellular-reduced glutathione (GSH) concentration. Water-soluble protein (WSP) from broad beans scavenges free radicals. The effects of WSP on the glutathione system were examined in PDL 20 (early passage) and PDL 50 (late passage) human lung fibroblasts (TIG-1). Methods: To determine cytosolic glutathione peroxidase (GSH-Px) activities, glutathione reductase (GR) activities, oxidized glutathione (GSSG) concentrations, and GSSG/reduced glutathione (GSH) ratios, WSP and hydrocortisone (HC) treatments of TIG-1 cells (PDL 20→50 and PDL 50→75) were performed for 40 days. We also investigated the GSSG concentrations and GR activities in PDL 20 cells that were continuously treated with WSP until PDL 39 and 55. Results: GSSG concentrations decreased in WSP- and HC-treated PDL 50→75 cells. The GSSG/GSH ratios in PDL 50→75 cells became low after the treatments. Increases in GR activities were observed in treated PDL 50→75 cells. The decline in the GSSG concentration of PDL 50→75 cells correlated with the increase in GR activity. The GSSG levels in control cells were higher following cellular age, whereas the levels in treated cells were lower than those in the control. The studies on cellular age-related changes indicated that greater increases in GR activity were found in treated cells than in the control. Conclusion: These results indicated that WSP influences the GSSG concentration that is associated with cellular aging, but the mechanism of GSSG reduction by WSP remains unknown. The enhancement of glutathione status following WSP treatment may be related to the delay in the cellular aging.

Effect of Qingfei Oral Liquid on Tumor Necrosis Factor-? mRNA gene expression of human embryonic lung fibroblasts induced by adenovirus Type3I,7b / 中华中医药杂志

Objective:To study the effect ofQingfei Oral Liquid medicated serum on the tumor necrosis factor-?(TNF-?) mRNA gene expression ofhuman embryonic lung fibroblasts induced by adenovirus Type3I, 7b.Methods:We determined the TNF-? mRNA ofADV-infected human embryonic lung fibroblasts before and after adding the medicated serum by in situ hybridization.Results:Adenovirus could up-regulate the TNF-?mRNA ofhuman embryonic lung fibroblasts(P

Inhibitory effect of laminarin (acidic polysaccharide) J201A on the proliferation of Human Lung Fibroblasts and its related mechanism of action / 中国海洋药物

To study the inhibitory effects of laminarin (acidic polysaccharide) J201A on the proliferation of human lung fibroblasts (HLF) and its related mechanism of action. Methods: Effect of J201A on the proliferation of HLF was evaluated by MTT assay, and the effects on cell cycle and synthesis of proteins of HLF were assessed by flow cytometry. The existence of J201A receptors on HLF was confirmed by fluorescent staining assay. Results:J201 A inhibited the synthesis of proteins and the proliferation of HLF at G0/G1 stage of cell cycle and J201A receptors existed on HLF. Conclusion:J201A exerted its antifibrotic activity by inhibiting HLF at G0/G1 stage of cell cycle and the synthesis of proteins.

Nickel Compound-induced DNA Damage in Human Lung Fibroblasts / 环境与健康杂志

Objective To observe the DNA damage in the human lung fibroblasts(HLF)induced by insoluble nickel compounds(Ni3S2 and Ni2O3)and soluble nickel compound(NiSO4),and to study the mechanism of carcinogenesis.Methods The DNA damage of HLF cells treated with different concentrations of Ni3S2,Ni2O3 and NiSO4 were detected with the method of single cell gel electrophoresis(SCGE).Results All of the three kinds of nickel compounds significantly caused DNA damage in HLF cells,the average tail moments of all nickel-treated groups were larger than those in the control group(P