Peroxisome Proliferator Activated Receptor-gama Inhibit Lung Fibrosis In Connective Tissue Disease-interstitial Lung Disease Research

BackgroundThe clinical manifestations of connective tissue disease(CTD) in lungs has a variety of performance, which can involve the airways, pulmonary interstitial lung parenchyma, vascular and pleura. Interstitial lung disease (ILD) is the main complications of the CTD when the lung is related. Pulmonary ILD also known as diffuse parenchymal lung disease, which has a common imaging, pathology and similar clinical manifestations. The mobility of ILD is up to25%, but10years survival rate is only60%. Hormones and immunosuppressive therapy are commonly used methods to treat the disease, But both of the treatment is ineffective, furthermore the adverse reactions of them limit the medicine’long term application. Therefore, there is an urgent need to detect the effective method to treat the disease. The main pathological change of ILD is alveolar chronic inflammation, the interstitial cell excessive hyperplasia and extracellular matrix deposition. There is a large number of pro-inflammatory and pro-fibrotic cytokines participating in the process of lung fibrosis. TGFβ1, a well-known proinflammation cytokine, has been confirmed as one of the most critical fibrogenic cytokines, because of its special function that promoting cell chemotaxis, proliferation and differentiation, and extracellular matrix synthesis and adhesion, reducing extracellular matrix degradation. Rrecent years, experiment, in vitro and in vivo, confirmed the peroxisome proliferator-activated receptor y (peroxisome proliferators activated receptor gamma, PPARy) can delay the process of pulmonary fibrosis promoted by TGFβ1pathway. The exact mechanism of inhibition is unknown. In this study, we will primary culture human lung fibroblasts.. and check the change of a-SMA, type I collagen, CTGF mRNA acetylation Smad3in fibroblast treated by TGFβ1and rosiglitazone to observe the effect of antifibrotic role and its mechanism.Objective1.Compare the different expression of PPARy in the lung of CTD-ILD patient and normal person to analysis the possible role of PPARy in the process of pulmonary fibrosis.2.Primary culture lung fibroblasts to understand the impact of TGFβ on cell differentiation and to observe whether PPARy ligands has inhibitory effect on TGFβ-induced cell differentiation.3.To explore whether the mechanism of PPAR on cell differentiation induced by TGFP is to combine with Smad3competitively.Materials and Methods1.1The study inclusion criteriaSpecimen were collected from patients with rheumatoid who take the CT-guided fine-needle aspiration lung biopsy from December2000to December2010, in the Guangdong Provincial People’s Hospital.37patients were included with29females and8males, mean age47.36±2.24. Including SSC6cases, SLE5cases, PSS5cases, PM/DM10patients, RA,3cases, vasculitis4cases, MCTD4cased. Normal lung tissue from20lung cancer patients, don’t smoke and no infection, was chosen as control, including12females and8males, mean age was45.62±1.12. The difference of age and gender between experimental and control group was not statistically significant.CTD-ILD inclusion criteria:meet the following①+⑤±any items.①meet the common international diagnostic standard to diagnosis various types of connective tissue disease;(2)cough, sputum, chest tightness, chest pain, and shortness of breath and other respiratory performance, exclude obstructive ventilatory disorders;③lung function:restrictive ventilatory dysfunction, gas diffusion function is reduced;④image changes:high-resolution CT (HRCT) examination showed ground-glass grid, honeycomb-like, fibrous cord-like shadow and cystic degeneration.⑤lung biopsy performance:in line with the2002American College of Chest Science and the European Respiratory Society (ATS/ERS) develop pathological manifestations, and to exclude pregnancy, infections, drugs, cancer and smoking and other factors. The study was approved by the local ethics committee, and to obtain the informed consent of all participants1.2Experimental Methods1.2.1Immunohistochemical:Paraffin-embedded lung tissue were sectioned at5-μm thickness then SP method was used to take the immunohistochemistry stainning. In short:after dewaxing, EDTA(PH=9.0)was usd to repair the antigen in the pressure cooker, when the slides were cooled down to room Temperature, then rabbit anti human PPARy (CST1:200) were incubated for60min at room temperature, and the negative control was replaced by PBS. the enzyme-labeled anti-rabbit secondary antibody (gene Company) were added and incubated at room temperature for30min, the benzidine ammonia (DAB)was used to stain the positive signal. After staining five400times field were randomly selected to count the positive ratio using the IPP software.1.2.2Cell culture:Select normal lung tissue adjacent with cancer from cancer patient whose age was less than55years, and no infection, no-smoking patients for primary fibroblasts cell culture. The lung fibroblasts were cultured in DMEM medium[4.5g/L glucose,10%fetal bovine serum (FBS),0.1%penicillin/streptomycin and glutamine] at37℃in5%CO2incubator. Flow cytometry vimentin to determine the lung fibroblast cell types.4to10passage of fibroblast cells were used as experimental subjects.1.2.3MTT method for cell activity:Cells were planted in96-well plates at the concentration of5×104cells per well. TGFβ1and rosiglitazone were added in the well when cell was in the exponential growth phase, the concentration of rosiglitazone was (1,5,10,20, and40μmol/L), and blank control was setted.72hours later in37℃containning5%CO2incubator20μl MTT was added. Conational culture for4hours.200μl dimethyl sulfoxide (DMSO) was added for another30min to terminate the culture. Measure the absorbance value at560nm.1.2.4Western blot:(1) lung fibroblasts cells were planted in6-well plates at the concentration of1×105-1×106per well. on six-well plates, cultured in serum-free medium for12hours. Then replace medium with DMEM medium containing10%serum.2hours before adding TGFβ1(5ng/ml), different concentrations of rosiglitazone were added, The group were divided into blank group, TGFβ1(5ng/ml) group and TGFβ1(5ng/ml)+rosiglitazone group (1,5,10,20and40μmol/L), cultured for48h, whole-cell protein were extracted.(2) Lung fibroblasts cells were planted in6-well plates at the concentration of1×105-1×106per well. cultured in serum-free medium for12hours. Then replace medium with DMEM medium containing10%serum. The cells were divided into blank group, TGFβ1(5ng/ml) group and TGFβ1(5ng/ml)+rosiglitazone group (20μmol/L), TGFβ1(5ng/ml)+rosiglitazone+GW9662(10μmol/L) group. BCA method was used to measure the concentration of protein. The same amount (10～15μg) protein were used for western blot.5%to10%polyacrylamide gel were used for electrophoretic separation, after the electroporation, and skim milk powder, polyvinylidene difluoride (PVDF) membrane were incubated at4℃after night with an anti-a-SMA (1:400Boshide), Then the membrane were incubated with goat anti-rabbit IgG secondary antibody (1:2000) for1hour. Enhanced chemiluminescence (ECL) was used for detect the signal. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal experiment. The experiment was repeated three times. Image J software was used for measuring the gray value.1.2.5RT-PCR:the mRNA sequence of type I collagen, CTGF, PPARy and GAPDH were inquiried at GenBank of NCBI Website,the mRNA were design and synthesis by Jierui biotechnology company. GAPDH:sense-5’-GCTGATGATCTTGAGGCTGTTGTC-3’; antisense-5’-CGCTGAGTACGTCGTGGAGTC-3’; CTGF:sense5’-TCGGGAAGGGGCAGTCAGGGCT-3’; antisense-5’-CCAACCGCAAGATCGGCGTGTG-3’; COLIA:sense-5’-CTTGGTCGGTGGGTGACTCTG-3’; antisense-5’-GGCAAGGTGTTGTGCGATGAC-3’. The harvest pretreated lung fibroblasts extracted total RNA using Trizol law with reverse transcriptase kit containing reverse transcriptase PrimeScriptTMRTase (Dalian TaKaRa). The RNA was reverse transcribed into cDNA and stored at-20℃. RNA by reverse transcription and quantitative real-time PCR amplification and specific reference to the kit instructions. Data processing:the calculation of the mRNA expression of2-ΔΔCT, ΔCT=CT (experimental group-the control group).1.2.6Immune co-precipitation:detected by Western blot acetylation of Smad3expression and the conjunction of Smad3or PPARgamma with P300:lung fibroblasts cells were planted in6-well plates at the concentration of1×105-1×106per bottle. Then following conditions:(1)60,90,180min after induction by TGFβ1(5ng/ml), cells were harvested and total protein were extracted, co-immunoprecipitation was used to detect the change of acetylated Smad3;(2)Adding rosiglitazone(20μmol/L) and(or) TGFβ1(5ng/ml) in fibroblasts. Dividing cells into blank group, the TGFβ1(5ng/ml) group, rosiglitazone (20μmol/L) group and TGFβ1+rosiglitazone group. After culture for90min, total protein was extract by lysate and detecting the combination of P300with Smad3and PPARy. The precipitate Antibodies of Smad3and P300(1:100CST Company) were added to the pre-processed in (1)(2) conditions for90min at4℃. Added to the washed Protein G/A agarose (15ul/lug antibody)40C after90min at4℃14000g for6s, centrifugal supernatant was discarded, the cell lysis buffer (without protease inhibitors) was added for4℃, and14000g for6seconds washed3times, the supernatant was removed. About20ul precipitation protein denaturant was placed in boiling water for5minutes,-20℃for storage.5%to10%polyacrylamide gel were used for electrophoretic separation, after the electroporation. and skim milk powder, polyvinylidene difluoride (PVDF) membrane were incubated at4℃after night with an anti-a-SMA (1:400Boshide), Then the membrane were incubated with goat anti-rabbit IgG secondary antibody (1:2000) for1hour. Enhanced chemiluminescence (ECL) was used for detect the signal. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal experiment. The experiment was repeated three times. Image J software was used for measuring the gray value.1.3Statistical methodstData processing were analyzed using SPSS13.0software. Results were expressed as mean±standard error of the mean. Statistical analysis of data was performed using one-way ANOVA, or further pairwise comparisons using LSD test; Non-normal distribution of the data with the [M (QL-QU)], multiple sets of data were compared using the Kruskal-Wallis H test and the Mann-Whitney analysis. Enumeration data using the chi-square test. p<0.05was considered statistically significantResults2.1.immunohistochemistryPPARy in the lung tissue is mainly expressed in nucleus of alveolar epithelial cells, fibroblasts, macrophages cells. In the37cases of patients with CTD-ILD, the expression of PPARy is markly higher than20normal cases [0.92%(1.44%),3.50%(1.94%)],(Z=-8.92, P<0.01)。2.2.Cell activityMTT assay showed that the Absorbance values of different concentrations of PPARy ligands rosiglitazone (0,1,5,10,20,40μmol/1) were (0.35±0.21,0.34±0.34,0.27±0.17,0.19±0.07,0.33±0.36VS0.23±0.09). Results showed that there is no difference among of them. So we conclude that the decrease of a-SMA and collagen type I not due to the toxic of medicine itself but the effect of PPARy.2.3. TGFβ1promoting human lung fibroblasts differentiate into myofibroblast cells:Compared with the control group, the expression of a-SMA in lung fibroblast cells was increased in cells48hours treated by TGFβ1(0,1.25,2.5,5and7.5ng/ml) with a concentration-dependent manner, and reached an optimal stimulation effect when the concentration reached5ng/ml. In the1.25ng/ml group the difference was showed no significant difference (P>0.05), the changes of a-SMA expression in the rest groups were statistically significant (P<0.05, P<0.01, P<0.01)2.4PPARγ ligands could significantly inhibit TGFβ1-induced differentiation of human lung fibroblasts.Compared with the control group the expression of a-SMA was decreased relatively in the group treated by rosiglitazone (1,5,10,20and40μmol/L). In the rosiglitazone1μmol/L group, the difference of a-SMA’expression was no significant. While the difference in rosiglitazone concentrations (10,20and40μmol/L) group was significant compared with the control group (P<0.05, P<0.01, P<0.01, P<0.01).2.5The inhibition of rosiglitazone on TGFβ1-induced differentiation of human lung fibroblasts depends on PPARy.In human lung fibroblasts, rosiglitazone (20μmol/L) can inhibit the expression of a-SMA on TGFβ1(5ng/ml) induced human lung fibroblasts, but when adding GW9662(10μmol/L),a antagonists of PPARy, on the cells,the expression of a-SMA was returen to increase relatively, GW9662can offset most of the inhibition. The expression of a-SMA was decreased in the TGFβ1(5ng/ml)+rosiglitazone (20μmol/L) group(P<0.01). While the expression of a-SMA was significantly increased in TGFβ1+rosiglitazone+GW9662than the TGFβ1(5ng/ml)+rosiglitazone (20μmol/L) group (P<0.01)。2.6The inflution of rosiglitazone on the gene expression of type I collagen and the connective tissue growth factor:Compared to the control group, the MRNA level of type I collagen and CTGF was significantly decreased48hours after that the rosiglitazone (1,5,10,20and40μmol/L) was added (All P<0.05),2.7TGFβ1increased lung fibroblasts acetylation Smad3In human lung fibroblasts, co-immunoprecipitation-Western blot (IP-WB) testing was used to test acetylation Smad3at different time after TGFβ1(5ng/ml) was added. In lung fibroblasts induced by TGFβ1after60,90and180min, the expression of acetylated Smad3was increased and reached a peak at the90min, compared with the control group, the difference was statistically significant (P<0.05). 2.8Rosiglitazone by interfering smad3combination with P300to exert anti-fibrotic effectsThe combination of smad3and p300was increased in TGF compared with the control, while the combination was significantly decreased when the rosiglitazone was added. Interestingly the combination of PPAR y and P300was relatively increased compared with the TGFβ1group (P<0.05.DiscussionCTD-ILD is high clinical morbidity and mortality, and lacking effective treatment of interstitial lung disease, which has drawn increasing attention. The study found that PPARy has a variety of biological effects. It not only can promote fat and lipoprotein metabolism, regulating the body’s homeostasis, but also has a role in inhibiting the inflammatory response and anti-fibrosis. PPARy is ligand-activated. Therefore, we added thiophene exogenously the synthetic ligand rosiglitazone to understand the mechanism of PPARy anti-fibrosis effect in the lung tissue of patients with CTD-ILD and for detecting new therapeutic strategies.Our experiments also found PPARy protein expression was significantly reduced in the lung tissue of patients with CTD-ILD. PPARy is mainly expressed in alveolar epithelial cells, fibroblasts, macrophages, these cells are the major cellular components within the lung tissue, mainly involved in gas exchange, tissue repairment and inflammatory response. Macrophages can synthesize and release of lysosomal enzymes and fibrosing factor to stimulate fibroblast proliferation and collagen synthesis; epithelial cells of fibroblast proliferation and collagen synthesis regulation, but also through the basement membrane to reach the fibrotic lesions, the epithelium-the transformation process of the mesenchymal cells into myofibroblasts. Studies have found that PPARy can induce apoptosis of alveolar epithelial cells and inhibition of alveolar epithelial cells to mesenchymal transition, inhibition of monocyte chemoattractant protein-1, interleukin-8and to inhibit monocyte aggregates, reduce inflammation. Activation of PPARy can also inhibit fibroblast and myofibroblast proliferation, differentiation and extracellular matrix production. PPARy expression decreased to some extent, to accelerate the development of pulmonary fibrosis, so if we can improve the functional activity of PPARy, is likely to slow down the incidence of pulmonary fibrosis process development.Our previous have study found that play an important role in the development and progression of CTD-ILD, the experimental results showed that the PPARy ligands inhibited the induction of TGFβ1lung fibroblasts into myofibroblasts. MTT method confirmed that this effect is not PPARy ligand toxic effect itself. Myofibroblast secrete extracellular matrix components of I and III collagen. Similar to our study, the Milam and Genovese also found that PPARy ligand rosiglitazone can inhibit TGFβ1induced fibroblast proliferation, transformation, and improved bleomycin-induced lung injury in mic. But the TGFβ1pro-fibrotic effects of PPARy with inhibition mechanism is not clear.TGFβ1signaling pathway, TGFβ1activated TGFβ1receptor, the receptor is activated and promote catalytic Smad2/3phosphorylation, phosphorylated Smad2/3in combination with Smad4Smad2/3/4composite is formed, complexes are transferred by intracellular to the nucleus in conjunction with P300, Smad2/3/4/p300complex with Smad-specific binding components SBE (smad binding element, SBE) sequence is associated with regulating target gene expression, Smad3plays a critical role in this pathway. Enhanced activity of Smad3protein phosphorylation, acetylation or ubiquitination of histone modifications. PPARy activation with retinoid combination of the spatial conformation changes and capture coactivators P300formation of complexes binding to the peroxisome proliferator response elements (PPRE) in promoter region of target genes within the to cause target gene transcription. TGFβ1/Smad path and PPARy pathway shared in the cell activating factor P300, the P300may be a limited number of competition between the two pathways. The experimental results also show that the TGFβ1make the acetylation of Smad3protein expression in lung fibroblasts and the combination of Smad3and P300join PPARy ligand rosiglitazone, Smad3reduced in conjunction with P300. PPARy with antifibrotic role may compete with Smad3combined P300, makes Smad3/P300complex formation the reduced. Also exists in skin fibroblasts, the PPARy allotment body through the Smad-dependent role in the activation of transcription P300weakened collagen synthesis, making TGFβ1induced transcriptional effect of weakening.In summary, decreased expression of PPARy in the lung tissue of patients with CTD-ILD, in vitro experiments PPARy with the body was found to significantly inhibit TGFbetal-induced lung fibroblasts to myofibroblasts transformed, this role may compete with Smad3P300binding to achieve. Therefore, the.activation of PPARgamma with PPARgamma pathway is expected to become a new target for the treatment of connective tissue disease-related interstitial lung disease.Conclusion1. The expression of PPARy in the lung of CTD-ILD patients is reduced, which can not play a normal anti-inflammatory and anti-fibrosis function, leading to CTD-ILD occurrence and development indirectly.2. Rosiglitazone has anti-fibrotic effect, and is dependent on the PPARy.3.The mechanism of rosiglitazone’inhibition is achieved through the combination with P300competitively with Smad3.