| Pulmonary fibrosis is chronic lung disease caused by a variety of pathologic factors, its process is characterized by dysregulation of lung tissue injury and repair, reconstruction of extracellular matrix, excessive deposition of collagen, which can result in the damage of alveolar structure, ultimately leading to pulmonary fibrosis. The pathogenesis of pulmonary fibrosis has not been clarified clearly and lack of effective treatment. Arachidonic acid is released from phospholipids when activation of cell by stimuli. Arachidonic acid is converted to epoxyeicosatrienoic acid (EETs) by cytochrome P-450(CYP) epoxygenase, EETs are known to have diverse biological functions, such as diastolic vascular smooth muscle, lower blood pressure, reduce heart and brain ischemia reperfusion injury and anti-inflammatory antiapoptotic functions. However, EETs primary metabolic fate is conversion to dihydroxyeicosatrienoic acids (DHETs) by the soluble epoxide hydrolase (sEH) enzyme. It is not clear if EETs affect the pulmonary fibrosis. In this experiment we use specificity soluble epoxide hydrolase inhibitors (TPPU) inhibits sEH activity, which increase the beneficial actions of EETs. we First investigated the effect of EETs on bleomycin-induced PF in mice in the whole level. Then observed the effect of EETs on fibroblasts, further clarify EETs play a role in pulmonary fibrosis. Objective:The present study was designed to investigate the influence of EETs on bleomycin-induced PF in mice and proliferation、 differentiation and collagen synthesis of fibroblasts.Methods:(1) The model of pulmonary fibrosis in mice was established by intratracheal injection of bleomycin, Lung tissue was observed by HE and Masson staining;then collagen expression was detected by real-time PCR;(2) The influence of EETs on the proliferation of fibroblast was observed by MTT; the influence of EETs on the cell cycle of fibroblast was observed by flow cytometry;The effect of EETs on α-SMA、type Ⅰ and Ⅲ collagen mRNA expression in TGF-β1-induced fibroblast.Results:1. Animal experiment(1) the morphology changes in mouse lung tissue:HE staining showed:during BLM, fibroblast foci was formed for21th day, The extent of pulmonary fibrosis in TPPU+BLM group was significantly lower compared to BLM group; Masson staining showed:the collagen deposition in TPPU+BLM group significantly improvement than BLM group;(2) RT-PCR results showed that the mRNA expression of type Ⅰ collagen in TPPU+BLM group was significantly lower compared to BLM group (P<0.05). 2. Fibroblasts experiment(1) MTT results showed that pretreated with TPPU could inhibit the proliferation of fibroblasts in time-dependent manner (P<0.01)and in dose-dependent manner(0.1μM,1μM,10μM)(P<0.05);(2) Flow cytometry results showed that the ratio of S phase was significantly increased in TGF-β1group (24.5±1.05)%compared with normal group (17.24±2.12)%(P<0.05); compared with the DMSO+TGF-β1group, the ratio of S phase in TPPU(1μM)+TGF-(31group (20.37±2.6)%and TPPU(10μM)+TGF-β1group (19.36±0.13)%obviously reduced (P<0.05);(3) RT-PCR results showed that the expression of a-SMA mRNA in fibroblasts was very low and significantly increased after treat with TGF-β1(P<0.05); Pretreated with TPPU could obviously inhibit the expression of a-SMA (P<0.05);(4) RT-PCR results showed that the mRNA expression of Ⅰ、Ⅲ collagen in fibroblasts was significantly increased after TGF-β1stimulus compared with normal group (P<0.05); Pretreated with TPPU could attenuate the expression (P<0.05).Conclusion:1. TPPU could increase the concentration of EETs in lung,which alleviated the bleomycin-induced inflammatory and collagen deposition and inhibited the PF in mice; 2. EETs can inhibit the proliferation、differentiation and collagen synthesis of fibroblasts. |
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