Activation of macrophage peroxisome proliferator-activated receptor α inhibits macrophage inflammation-induced cardiac fibroblast activation and migration

AIM: To investigate the effect of macrophage peroxisome proliferator-activated receptor α (PPARα) activation on macrophage inflammation-induced activation and migration of cardiac fibroblasts. METHODS: Mouse bone marrow-derived macrophages were treated with vehicle, PPARα agonist WY14643 (10 μmol/L), angiotensin Ⅱ (Ang II; 1 μmol/L) or Ang II+WY14643 for 24 h, and the supernatants were collected as conditioned medium (CM) to stimulate cardiac fibroblasts for additional 24 h. The mRNA levels of PPARα, interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in the macrophages as well as fibrotic markers collagen type Ⅰ alpha 2 chain (Col1a2), collagen alpha 1 chain (Col3a1) and actin alpha 2 (Acta2) in the cardiac fibroblasts were detected by RT-qPCR. The protein levels of IL-6 and IL-1β in the macrophages as well as collagen I, collagen III and α-smooth muscle actin (α-SMA; encoded by Acta2 gene) in the cardiac fibroblasts were determined by Western blot. Wound-healing assay was applied to eva-luate the migration ability of cardiac fibroblasts. RESULTS: Ang II significantly increased the mRNA levels of pro-inflammatory factors, such as IL-6, IL-1α and TNF-α, but decreased the mRNA level of PPARα in the macrophages. Administration of PPARα agonist WY14643 dramatically decreased Ang II-induced mRNA levels of IL-6, IL-1β and TNF-α in the macrophages, and significantly decreased Ang II-induced protein expression of IL-6 and pro-IL-1β in the macrophages. The CM from Ang II-treated macrophages significantly up-regulated the mRNA levels of Col1a2, Col3a1 and Acta2 in the cardiac fibroblasts, which were inhibited by the CM from WY14643-treated macrophages. The same results were observed in the protein levels of collagen I, collagen III and α-SMA in the cardiac fibroblasts. Moreover, the CM from Ang II-treated macrophages significantly promoted cardiac fibroblast migration, whereas the CM from WY14643-treated macrophages markedly inhibited macrophage inflammation-induced cardiac fibroblast migration. CONCLUSION: WY14643-activated PPARα inhibits activation and migration of cardiac fibroblasts by attenuating Ang II-induced macrophage inflammatory response.     

Effect of recombinant interleukin-13 on 3T3 fibroblasts in vitro

AIM: To investigate the influence and mechanism of recombinant interleukin-13 (rIL-13) on fibroblasts. METHODS: 3T3 fibroblasts were divided into two groups: the treated group was treated with rIL-13 (80 μg/L, 24 h or 48 h) and the control was without rIL-13 treatment. Transmission electron microscope and Hoechst kit were used to observe morphology of 3T3 fibroblasts in both groups. The activity of proliferation in both groups was investigated and compared by MTT means. Western blot was used to analyze the level of collagen type I induced by rIL-13 in fibroblasts. The levels of IL-6 and IL-8 in the culture supernatants were determined by radioimmunoassay. RESULTS: The more ribosomes and mitochondrions, as well as bigger nuclei were found in the treated group. The production of IL-6 and IL-8, and proliferation ratio of fibroblasts treated with rIL-13 for 24 h or 48 h were increased obviously, compared with the control (P<0.01). The expression of collagen type I protein in treated groups was also significantly higher than that in control (P<0.01). CONCLUSION: rIL-13 upregulates the proliferation of fibroblasts. rIL-13 also has an effect on the production of proinflammatory factors and collagen type I. Taken together, these results suggest that IL-13 may play important roles in the pathogenesis of tissue fibrosis.     

Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cellsin vitro

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro.METHODS:Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p 53, bcl- 2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS:(1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic"ladder"of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p 53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10⁵ U/L),TNF-α(4×10⁵ U/L)and /or IL-1β (10×10⁴ U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group (P＜0.01).CONCLUSION:Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.     

Role of caveolae in high glucose-induced extracellular matrix production in rat mesangial cells

AIM：To investigate the role of caveolae in high glucose (HG)-induced extracellular matrix (ECM) production in rat mesangial cells (MCs). METHODS：Synchronized rat MCs were divided into normal glucose group, HG group, HG+methyl-β-cyclodextrin (β-MCD) group and HG+β-MCD+cholesterol (Chol) group. Western blotting was used to detect the protein expression of caveolin-1 (Cav-1), phosphorylated caveolin-1 (p-Cav-1-Y14) and collagen type 1 (Col I). The mRNA expression of Cav-1 was determined by real-time PCR. ELISA was used to measure the level of fibronectin (FN) in the supernatant. RESULTS：High glucose significantly increased the expression of FN and Col I. In HG 12, 24 and 48 h groups, the mRNA and protein levels of Cav-1 were not significantly different from those in HG 0 h group, whereas the level of p-Cav-1-Y14 was significantly increased. β-MCD significantly attenuated HG-induced elevation of p-Cav-1-Y14 and FN production, but had no effect on HG-induced Col I expression. All these responses to β-MCD were abolished by Chol. CONCLUSION：High glucose significantly increases the production of Col I and FN in rat MCs. FN production induced by high glucose is mediated by p-Cav-1-Y14.     

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.     

Effects of Jiedu-Qingfei mixture on NF-κB and p38 MAPK pathways in lung tissues of rats with Mycoplasma pneumoniae infection

AIM: To observe the therapeutic effect of Jiedu-Qingfei mixture on Mycoplasma pneumoniae (MP)-infected rat lung tissues and to explore its mechanism. METHODS: SD rats (n=40) were randomly divided into 4 groups:blank control group, model group, Jiedu-Qingfei group and positive control group, with 10 rats in each group. The rats in experimental groups were slowly dripped with 1×10⁹ CFU/L MP solution into their nostrils for 4 d. One rat in each group was sacrificed for MP nucleic acid detection at the second day after inoculation, and the other rats were given gavage therapy. The rats in blank control group and model group were intragastrically given the same volume of normal saline, the rats in Jiedu-Qingfei group were given 8 mL/kg Jiedu-Qingfei mixture daily for 4 weeks, and the rats in psoitive control group were given dexmethasone sodium phosphate (0.5 mg·kg^-1·d^-1). After the experiment, the rats were killed. The serum and bronchoalveolar lavage fluid (BALF) were collected for detecting the levels of interleukin-12 (IL-12), IL-13 and TNF-α by ELISA. The right lung tissues were used for pathological observation and HE staining, while the left lung tissues were used to detect the expression of NF-κB p50, I-κBα and p38 mitogen-activated protein kinase (p38 MAPK) at mRNA and protein levels. RESULTS: The results of MP nucleic acid detection showed that all the rats except blank control group were MP nucleic acid positive, indicating that the rat model of MP infection was successfully established. On the 1st day of the treatment, the pathological scores of the lung tissues in model group and Jiedu-Qingfei group were significantly higher than those in blank control group (P<0.05). After treatment, the pathological scores of the lung tissues in mo-del group were significantly higher than those in blank control group and Jiedu-Qingfei group. The levels of IL-12 in the serum and BALF in model group were significantly lower than those in blank control group after MP infection (P<0.05), while those after treatment with Jiedu-Qingfei mixture were significantly higher than those in model group (P<0.05). The levels of IL-13 and TNF-α in the serum and BALF of MP-infected rats were increased significantly, while those after treatment with Jiedu-Qingfei mixture were significantly lower than those in model group (P<0.05). The mRNA expression levels of NF-κB p50 and p38 MAPK in model group were increased significantly (P<0.01). After treatment, the mRNA expression levels of NF-κB p50 and p38 MAPK were decreased significantly compared with model group (P<0.01). The mRNA expression level of I-κBα in model group was significantly lower than that in control group. After treatment, the mRNA expression of I-κBα in Jiedu-Qingfei group was significantly higher than that in model group (P<0.05). The protein levels of NF-κB p50 and p38 MAPK in the lung tissues of model group were significantly higher than those of blank control group. After treatment, the protein expression of NF-κB p50 and p38 MAPK was decreased significantly. The protein level of I-κBα in model group was significantly lower than that in blank control group, and after treatment with Jiedu-Qingfei mixture, the protein expression level of I-κBα was increased significantly (P<0.05). CONCLUSION: Jiedu-Qingfei mixture may attenuate lung tissue inflammation caused by MP through NF-κB and p38 MAPK pathways.     

Effect of recombinant MIF on lung fibroblast proliferation and collagen synthesis in vitro

AIM: To investigate the influence and mechanism of recombinant macrophage migration inhibitory factor (rMIF) on fibroblasts. METHODS: MRC-5 fibroblasts were divided into two groups: the treated group was treated with rMIF (25-100 μg/L, 12 h, 24 h or 48 h) and the control was non-rMIF treatment. The activity of proliferation in both groups was investigated and compared by CCK-8 means. Synthesis of collagen in the culture supernatants was detected by the hydroxyproline. The expression of collagen type I mRNA was examined using RT-PCR analysis. The level of collagen type I protein induced by rMIF was quantified by Western blotting. RESULTS: The production of proliferation ratio of fibroblasts treated with 50 μg/L and 100 μg/L rMIF at 24 h or 48 h were increased obviously (P<0.05 or P<0.01). The collagen synthesis significantly increased after stimulation with 100 μg/L rMIF for 48 h (P<0.01). rMIF significantly increased the expression of collagen type I mRNA and protein in a dose dependent manner compared with control (P<0.05 or P<0.01). CONCLUSION: rMIF upregulates the proliferation of fibroblasts and has an effect on the production of collagen. These results suggest that MIF may play important roles in the pathogenesis of airway remodeling.     

Effect of angiotensin-(1-7) on angiotensin II-induced rat renal interstitial fibroblast activation

AIM: To explore the influence of angiotensin-(1-7) on angiotension II (Ang II)-induced activation and extracellular matrix secretion in rat renal interstitial fibroblasts (NRK-49F cells). METHODS: The NRK-49F cells were maintained and sub-cultured, then the cells were divided into control group, Ang II group, Ang-(1-7) group and Ang II+Ang-(1-7) group. The expression of α-smooth muscle actin(α-SMA),transforming growth factor β₁ (TGF-β₁) and insulin-like growth factor I(IGF-I) was detected by the method of immunocytochemistry when the cells were cultured for 72 h. The content of TGF-β₁, IGF-I and collagen type I(Col I) in the cultured supernatants were measured by ELISA. RESULTS: In control group and Ang-(1-7) group, only basic expression of α-SMA and almost no expression of TGF-β₁, IGF-I and Col I were observed. Compared with control group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was increased in Ang II group. Compared with Ang II group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was significantly decreased in Ang II+Ang-(1-7) group.CONCLUSION: Ang-(1-7) inhibits the activation of renal interstitial fibroblasts and decreases the Ang II induced secretion of Col I by suppressing TGF-β₁ and IGF-I expression.     

Intervention effects of apyrase on silica-induced pulmonary fibrosis in mice

AIM: To investigate the effect of apyrase on the experimental silicosis. METHODS: C57BL/6 male mice were randomly divided into control group, silica treatment group, silica+apyrase group and silica+NS group. A mouse model of lung fibrosis was induced by crystalline silica particles (50 mg/kg, via oropharyngeal instillation), and were sacrificed at 3 h, 7 d, 14 d and 28 d. Apyrase was delivered by oropharyngeal aspiration at the same time and 4 h after silica challenge. The lung indexes were calculated and the concentration of ATP was detected by bioluminescent assay. The mRNA expression levels of collagen type Ⅰ(Col Ⅰ), collagen type Ⅲ (Col Ⅲ) and transforming growth factor β1 (TGF-β1) were examined by real-time PCR. The protein levels of TGF-β1 in bronchoalveolar lavage fluid were measured by ELISA. RESULTS: The elevated lung index and collagen levels showed that silicosis model was established successfully. Compared with silica group, apyrase treatment significantly alleviated silica-induced inflammation, reduced inflammation score on day 7, and decreased the lung index, collagen volume fraction and the mRNA expression of Col Ⅰand Col Ⅲ on day 28. Treatment with apyrase effectively down-regulated the mRNA levels of TGF-β1 in the lung tissues and TGF-β1 protein levels in bronchoalveolar lavage fluid on day 7.CONCLUSION: Apyrase attenuates the pulmonary inflammation and fibrosis of silicosis, which may be related with down-regulation of ATP and TGF-β1 in the lung tissues.     

Effect of Yiqi Huayu Huatan decoction on expression of KLF15, HMGB1, NF-κB and its downstream inflammatory factors in rats with UUO-induced renal interstitial fibrosis

AIM: To observe the effect of Yiqi Huayu Huatan decoction (YHHD) on unilaterral ureteral obstruction (UUO)-induced renal interstitial fibrosis in rats, and to investigate the possible mechanism. METHODS: Female SD rats (n=48) were randomly divided into sham group, model group, telmisartan group, and low-, middle-and high-dose YHHD groups, with 8 rats in each group. The UUO model rats was established by ligating left ureter. The rats in sham group and model group were treated with equal volume of normal saline, others were treated with the corresponding drugs daily. After 12 weeks, the rats were sacrificed. The serum samples were collected for determining the concentrations of cystatin C (Cys-C) and uric acid (UA). The morphological changes of the renal tissue were observed by PAS staining. The collagen fiber was observed by Masson staining. The mRNA expression of Krüppel-like factor 15 (KLF15), high-mo-bility group box protein 1 (HMGB1), nuclear factor-κB (NF-κB), IκB, monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), fibronectin (FN), collagen type I (Col I) and Col-Ⅳ was detected by real-time PCR. The protein expression of KLF15, HMGB1 and NF-κB was detected by Western blot. The protein expression of MCP-1 was determined by the method of immunohistochemistry. RESULTS: Compared with sham group, the deposition rate of collagen fibers and the concentration of Cys-C in model group were significantly increased (P<0.05), the mRNA and protein expression of KLF15 was significantly down-regulated (P<0.05), while the mRNA expression of HMGB1, NF-κB, IκB, MCP-1, IL-1β, TNF-α, FN, Col I and Col Ⅳ and the protein expression of HMGB1, NF-κB and MCP-1 were significantly up-regulated (P<0.05). Compared with model group, the deposition rates of collagen fibers in middle-and high-dose YHHD groups and telmisartan group were significantly decreased (P<0.05), with down-regulated protein expression of HMGB1 and NF-κB and mRNA expression of IL-1β and TNF-α (P<0.05). The protein expression of KLF15 was significantly up-regulated in high-dose YHHD group and telmisartan group (P<0.05), while the protein expression of MCP-1 and the mRNA expression of FN were significantly down-regulated (P<0.05). The mRNA expression of KLF15 was significantly up-regulated in high-dose YHHD group (P<0.05), while the mRNA expression of MCP-1, Col I and Col IV was significantly down-regulated (P<0.05). The mRNA expression of NF-κB and IκB was significantly down-regulated and the concentration of Cys-C was significantly decreased in each dose of YHHD groups and telmisartan group (P<0.05). No significant difference of UA level among the groups was observed. CONCLUSION: YHHD alleviates renal interstitial fibrosis in a dose-dependent manner, and YHHD at high dose shows the most obvious effect. The mechanism may be associated with the up-regulation of KLF15 and the down-regulation of HMGB1, NF-κB and its downstream inflammation-related factors in the renal tissue.     

Effect of salvianolic acid B on high glucose-induced phenotypic transition and extracellular matrix secretion in human glomerular mesangial cells

AIM:To investigate the effect of salvianolic acid B (Sal B) on high glucose-induced phenotypic transition and extracellular matrix (ECM) secretion in human glomerular mesangial cells (HGMCs) and the underlying mechanisms. METHODS:HGMCs were randomly divided into control group, high glucose group and high glucose plus high dose, medium dose and low dose of Sal B groups. The HGMCs except those in control group were exposed to high glucose (33.3 mmol/L) for 72 h, while those in Sal B groups were co-incubated with indicated concentrations of Sal B. The protein levels of α-smooth muscle actin (α-SMA), transforming growth factor-β₁ (TGF-β₁) and phosphorylated Smad2 and p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. The secretion levels of collagen type I (Col I), collagen type Ⅲ (Col Ⅲ), fibronectin (FN) and laminin (LN) were measured by ELISA. RESULTS:Exposure to high glucose markedly increased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs (P<0.01). The phosphorylation levels of Smad2 and p38 MAPK were also significantly increased (P<0.01). Co-incubation with Sal B evidently decreased the protein expression of α-SMA, TGF-β₁, Col I, Col Ⅲ, FN and LN in the HGMCs induced by high glucose (P<0.05 or P<0.01). The phosphorylated levels of Smad2 and p38 MAPK were also reduced noticeably (P<0.05 or P<0.01). CONCLUSION:Sal B significantly suppresses high glucose-induced phenotypic transition and ECM secretion in the HGMCs, which might be attributed, at least partly, to inhibition of TGF-β₁/Smad signaling pathway and p38 MAPK activation.     

Effect of CD151 on biological characteristics of human umbilical cord mesenchymal stem cells

AIM：To study the effect of CD151 on the biological characteristics of human umbilical cord mesenchymal stem cells (hUC-MSCs). METHODS：CD151 expression on hUC-MSCs was interfered by siRNA. The cells were divided into siRNA-CD151 group and negative control group (treated with siRNA-NC). The efficiency of interference after 72 h and the changes of other surface markers were detected by flow cytometry. The ability of differentiation was assessed by oil red O and von Kossa staining. The cell cycle was analyzed by flow cytometry. The mRNA expression of CD151, hepatocyte growth factor (HGF), transforming growth factor β1 (TGF-β1), cyclooxygenase 2 (COX-2) and indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs was detected by real-time PCR. The secretion of HGF by hUC-MSCs was measured by ELISA. RESULTS：The results of flow cytometry showed that the expression of CD151 (11.97±2.63 vs 95.66±1.56, P<0.01) and CD105 (93.66±0.21 vs 83.37±0.71, P<0.05) on hUC-MSCs in siRNA-CD151 group was lower than that in negative control group. The consistent results were also achieved by using the method of real-time PCR. Treatment with siRNA-CD151 down-regulated the progress of the cell cycle as the G1 phase increased and the S phase decreased. The mRNA expression levels of HGF and TGF-β1 in hUC-MSCs in siRNA-CD151 group were lower than those in negative control group, and opposite result of COX-2 mRNA expression was observed. The IDO mRNA in hUC-MSCs was unchanged with IFN-γ stimulation for 24 h. HGF concentration in siRNA-CD151 group was decreased as compared with negative control group. CONCLUSION：Interfering CD151 expression on hUC-MSCs doesn’t change other surface markers except CD105, and maintains the capacity of adipogenic differentiation. However, it changes the osteogenic differentiation, proliferation and the expression of immunomodulatory cytokines.     

Effect of decorin on proliferation and collagen type I synthesis of stiff knee joint synovial type B cells

AIM: To explore the effects of decorin on procollagen type I (PcI), mRNA expression,collagen type I synthesis and proliferation of synovial type B cells of stiff knee joint synovial membrane. METHODS: Type B cells of synovial membrane were isolated from the stiff knee joint synovial membrane and cultured in vitro. The cells were treated with decorin at concentrations of 0.1 mg/L, 5 mg/L and 10 mg/L. After cultured for 24 h, 48 h and 72 h, the cell proli-feration rates were measured by MTT colorimetric determination. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The mRNA level of Pc I was detected by RT-PCR, while collagen type I was measured by Western blot. RESULTS: The proliferation of synovial type B cells was significantly inhibited, the percentage of synovial type B cells at G₁ phase was significantly increased by 5 mg/L and 10 mg/L decorin (P<0.05), and PcⅠmRNA expression and collagen type I synthesis were significantly decreased. The cells with late apoptosis were not found in control group and experimental groups. CONCLUSION: Recombinant human decorin inhibits synovial type B cell proliferation and decreases PcⅠmRNA expression and collagen type I synthesis in synovial type B cells of stiff knee joint synovial membrane in vitro, suggesting that decorin potentially contributes to the therapy of human knee stiffness.     

Effects of bilberry anthocyanins on MMP2 and collagen I in human fetal scleral fibroblast in vitro

AIM:To investigate the effects of bilberry anthocyanins on matrix metalloproteinase 2 (MMP2) and collagen type I (collagen I) expression in human fetal scleral fibroblasts (HFSF) in vitro and to provide experimental data for the prevention and treatment of myopia by bilberry anthocyanins. METHODS:HFSF were treated with bilberry anthocyanins at different concentrations (0, 10^-6, 10^-5, 10^-4, 10^-3, 10^-2, 10^-1 and 1 g/L). The effects of bilberry anthocyanins on the viability of HFSF for different time (6 h, 8 h, 12 h, 24 h and 48 h) were measured by MTT assay. The cell cycle distribution and apoptosis of HFSF with highest viability (10^-1 g/L bilberry anthocyanins for 12 h) were analyzed by flow cytometry. The mRNA expression of MMP2, COL1A1 and COL1A2 in the HFSF was detected by RT-qPCR. The protein expression of MMP2 and collagen I was determined by Western blot. RESULTS:The cell viability was the highest after treatment with bilberry anthocyanins at a concentration of 10^-1 g/L for 12 h. Compared with blank control group, 10^-1 g/L bilberry anthocyanin group showed increased cell numbers in S and G₂ phases (P<0.05), but no significant difference of the apoptotic rate was observed. The mRNA expression of MMP2 was decreased (P<0.05), and that of COL1A1 was increased (P<0.05). No significant difference of COL1A2 expression was seen. The protein expression of MMP2 was decreased (P<0.05), and collagen I protein was increased (P<0.05). CONCLUSION:Bilberry anthocyanins inhibit the expression of MMP2 and increase the expression of collagen I in the HFSF.     

Effects of arsenic trioxide on T-bet/GATA3 signal pathway in MRL/lpr mice

AIM: To investigate the effect of arsenic trioxide (ATO) on T-bet/GATA3 signal pathway in MRL/lpr mice.METHODS: MRL/lpr mice and C57BL/6J mice at the age of 20 weeks were chosen and then divided in 2 different sub-groups, respectively. The mice in 2 sub-groups received ATO (0.4 mg·kg^-1·d^-1) and sodium chloride (NS, volume weight-determined) by intraperitoneal injection respectively for 2 months. Afterward, the spleens were isolated from the MRL/lpr and C57BL/6J mice under pathogen-free condition and the suspensions were prepared. The mRNA level of T-bet, GATA3, IFN-γ,IL-4 and the mRNA ratio of T-bet/GATA3 were detected by RT-qPCR. The protein expression of T-bet and GATA3 was determined by Western blot. The serum levels of IFN-γ and IL-4 were measured by ELISA.RESULTS: The mRNA and protein levels of T-bet, IFN-γ and the mRNA ratio of T-bet/GATA3 in NS group of MRL/lpr mice were higher than those in NS group of C57BL/6J mice (P<0.05). However, the GATA3 and IL-4 were lower in NS group of MRL/lpr mice in both mRNA and protein level (P<0.05). In MRL/lpr mice, the mRNA and protein levels of T-bet, IFN-γ and the mRNA ratio of T-bet/GATA3 were lower in ATO group compared with NS group (P<0.05), no difference was found in GATA3 and IL-4. No difference of the indexes mentioned above between ATO group and NS group in C57BL/6J mice was observed.CONCLUSION: ATO may affect the signaling pathway of T-bet/GATA3 to down-regulate the mRNA expression and the protein secretion of IFN-γ by decreasing the expression of T-bet in MRL/lpr mice.     

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.     

Regulation of rat pulmonary fibroblasts differentiation into myofibroblasts though RhoA/ROCK pathway mediated by TGF-β1

AIM：To investigate the regulatory effect of RhoA/Rho-associated coiled-coil-forming protein kinase (ROCK) pathway mediated by transforming growth factor β1 (TGF-β1) on the differentiation of pulmonary fibroblasts into myofibroblasts. METHODS：Primarily cultured fibroblasts were obtained by trypsin digestion from the lung of neonatal rats. The fibroblasts were stimulated with TGF-β1 for different durations and were divided into control group, TGF-β1 induction group and Y-27632 treatment group. The distribution and expression of p-RhoA, ROCK, phosphorylated myosin binding subunit of myosin light chain phosphatase (p-MBS), serum response factor (SRF), α-smooth muscle actin (α-SMA),type I collagen and type Ⅲ collagen in the cells were detected by the methods of immunocytochemistry and Western blotting. RESULTS：A lot of parallel and cross arranged filaments labeled by α-SMA antibody appeared in the cells after TGF-β1 stimulation. The cultured cells stimulated with TGF-β1 were all myofibroblasts at 24 h determined by immunocytochemistry. The expression levels of p-RhoA, ROCK, p-MBS, SRF, α-SMA and type I and type III collagens were increased gradually with the extension of TGF-β1 stimulation time. The expression of RhoA/ROCK signaling protein in the cells stimulated with TGF-β1 (peaking at 6 h of exposure) was 2.96 folds higher as compared with the non-stimulated cells. The expression of SRF protein (peaking at 12 h of TGF-β1 exposure) was 4.55 folds higher as compared with the non-sti-mulated cells. The expression levels of α-SMA and type I and type III collagens (peaking at 24 h of TGF-β1 exposure) were 4.06 folds, 2.19 folds and 3.04 folds higher as compared with the non-stimulated cells, respectively. Compared with TGF-β1 induction group, the protein expression levels of ROCK, p-MBS, SRF, α-SMA and type I and type III collagens were significantly decreased at the corresponding time points in Y-27632 treatment group. CONCLUSION：TGF-β1 induces the differentiation of pulmonary fibroblasts into myofibroblasts, and then promotes the synthesis of collagen through the activation of ROCK pathway, which possibly plays an important role in the formation of pulmonary fibrosis.     

Effects of bone mesenchymal stem cell transplantation on silicosis fibrosis in rats

AIM: To study the effects of exogenous bone mesenchymal stem cell (BMSC) transplantation on silicosis fibrosis in rats, and to explore the dose-effect relationship. METHODS: BMSCs were isolated and cultured from male 5-week-old SD rats in vitro. Fifty healthy female SD rats were randomly divided into 5 groups: control group, silicosis model group, BMSCs treatment A group (1×10⁹ cells/L), BMSCs treatment B group (3×10⁹ cells/L) and BMSCs treatment C group (5×10⁹ cells/L). The silicosis model was made by one-time infusion of silica dust suspension using the non-exposed tracheal intubation, and different doses of BMSCs were given for intervention therapy. All the rats were sacrificed on the 21st day after the model was established. The morphological changes of the lung tissues were observed by HE staining and Masson staining. The localization and distribution of tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) were determined by the method of immunohistochemistry. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III were detected by Western blotting. The sex-determining region (SRY) protein was searched by an immunofluorescence method to confirm the homing of BMSCs. RESULTS: Compared with control group, the silicosis model group had significant alveolitis changes, silicon nodule formation, collagen deposition and other pathological characteristics. Compared with silicosis model group, the pathological changes in BMSCs treatment A group were improved. The conditions of BMSCs treatment B group were also improved significantly. However,the pathological changes in BMSCs treatment C group were increased obviously. The protein levels of TNF-α, TGF-β, collagen type I and collagen type III in the lung tissues ranked as follows: BMSCs treatment C group > silicosis model group > BMSCs treatment A group > BMSCs treatment B group > control group. The difference between BMSCs treatment C group and silicosis model group was not statistically significant, and the differences between the other groups were statistically significant. The SRY-positive cells were observed in BMSCs treatment B group, but no significant expression in the heart, liver, spleen and kidney tissues was observed. CONCLUSION: The exogenous BMSC transplantation antagonizes the development of silicosis fibrosis in rats, which has dose-effect relationship.     

Effects of sterigmatocystin on IL-2 and IFN-γ secretion and expression in murine spleen cells in vitro

AIM: To explore the effects of sterigmatocystin (ST) on IL-2 and IFN-γ expression and secretion in murine spleen cells in vitro. METHODS: The secretion and expression of IL-2 and IFN-γ in murine spleen cells after ST pretreatment at five different dosages(0.125 mg/L,0.25 mg/L,0.5mg/L,1 mg/L,2 mg/L) were studied with ELISA and semi-quantitative RT-PCR method, respectively. RESULTS: Pretreatment of murine spleen cells in vitro with ST at five different dosages affected the IL-2 and IFN-γ secretion at protein level and expression at mRNA level of the treated cells. The effects varied dependently to the ST dosage. At relatively lower dosages, ST induced the expression of IL-2 and IFN-γ in murine spleen cells, while at relatively higher dosages, inhibitory effects were found, with the most significant inhibitory effects seen in ST 1 mg/L group. CONCLUSION:ST affected the se-cretion and expression of IL-2 and IFN-γ in treated murine spleen cells.At relatively lower dosage, ST induced IL-2 and IFN-γ secretion and expression, while at relatively higher dosages，inhibitory effects appeared.