Experimental Study Of Curcumin Prevention And Treatment On Bleomycin-induced Pulmonary Fibrosis In Rats

Background & ObjectivePulmonary fibrosis(PF) is a kind of diffuse interstitial lung disease which caused bymany causes and has similar pathogenesis, Its pathogenic characterize is which alveolarstructure destruction and diffuse alveolitise in the early, then cause over aggradation of ECMand gaudily leading to fibrosis, consequently cause respiratory function destruction. Most ofthe patients died from respiratory failure from progressive pulmonary fibrosis. Itspathogenesis is not thoroughly understand till now, so recently the incidence rate and thedeath rate of pulmonary fibrosis has trend of increasing. The effects of traditional medicine ofGlucocorticoid and cell toxicant is dissatisfactory to treat pulmonary fibrosis, so at present,the urgent question affronting the medical science field is to seek effective medicine forpulmonary fibrosis.The research show that curcumin possess widespread pharmacological action inrecently years, for example anti-inflammatory, anti-oxidation, anti-ischemic, anticancer,inhibiting hepatic fibrosis. To discuss the preventing and treating effect of curcumin onpulmonary fibrosis, this experiment was adopted rat model induced by bleomycin (BLM), toexplore the effect and mechanisms of curcumin on PF from aspects of integral and molecularlevel in vivo and in vitro, which can provides experimental foundation for clinic therapy.Methods & Results1 Experimental studies in vivo1.1 Methods 144 male Sprague-Dawley rats were randomly divided into 6 groups (24 ratsin each group). Rats in the model control group, positive medicine group, and high, moderateand low curcumin groups were injected with a single dose of bleomycin by trachea, and ratsin sham-model control group with same volume normal saline. 1 day after the injection, oraladministration of drug daily until they were killed 1 day before. Curcumin solution ofdifferent dosages (200mg, 100mg, 50mg·kg^-1·d^-1) was respectively given to rats in the high,moderate and low curcumin group by gavages, while equal volume of normal saline wasgiven to those in the sham-model control group and model control group, and an equalvolume of prednisone(0.56mg·kg^-1·d^-1) was saline was given to those in positive medicinecontrol group.8 rats of each group were selected from every group on 7th day, 14th day and 28th day were be executed, 6 of them were made for light microscope specimen to fix. 2 ratswere operated to open thorax and get lung tissue speedily. The latter was put into liquidnitrogen promptly to be frozen for immunohistochemistry, western blot and making electronmicroscope specimen. Light microscope specimens were made in regularly day, which werestained with HE, Mallory staining for pathological diagnosis. The Immunohistochemiscalglass covers were dealt with DEPC for use. Transmission electron microscope specimenswere made with routine method. Method and steps of material getting for ruthenium redstaining electron microscope specimen were the same as that of transmission electronmicroscope specimens especially fixing with pre-fixer and post-fixer. Method of making ultrathin sections was the same as that of transmission electron microscope. The lung tissue wasdivided into didn't dye area, deep dye area and shallow dye area, image analysis was adoptedthe Image-Pro Plus 5.0 auto to calculate the area (mm²) of each part, and analyzed bySPSSll.5 software.1.2 Results Results with HE staining under light microscope:pulmonary tissue structure ofsham operation group of 7d, 14d and 28d were normal. Capillaries of lung were congested,the breadth of alveolus interval to increase and dropsy, and there are large numbers ofneutrophil and monocyte were observed, fiber hyperplastic focuses were observed lyingaround alveolus interval and bronchiole in model group of 7d; Exudation and pleurathickening were seen in positive control group that didn't exist in rest group; various degreesinflammation in the high medium and low curcumin groups of 7d. Except sham operationgroup the inflammation of every group of 14d were lighten and showed various degreesfibrosis focus. Inflammatory of every group reduced on 28d. A few inflammatory cellsremained in part of area. Fibrosis degree of model group was more obviously and had a lotconsolidation area, this showed the animal model replication was success. The condition ofpositive control group was better. Fibrosis degree was not too high in alveolus interval.Conditions of high, medium and low curcumin groups became good more distinctly. Fiberhyperplastic focus was less in alveolus interval and there weren't inflammatory cells inalveolus. Fibrosis degree around the bronchiole was obviously lower than that of model groupand positive control group. Among the 3 groups medium and high curcumin groups weremore effective because few little fibrosis focus remained and structure of alveolus wasrestored closely to regular comparison group. The results showed curcumin had clear curativeeffect to PF by reducing fiber piling and restraining forming of fibrosis.Mallory staining results: The results showed hyperplasia in alveolus interval of high,medium and low curcumin groups was not identified. The degree of fibrosis grade was verylow. Especially pathological changes degree of medium curcumin groups was lighter. Fibrosisgrade was low. Total collagen was obviously little.The image quantitative analyzing results showed: the model group and sham-modelcontrol group compare: the image quantitative analysis result and the pathology check consistent. The medium and low groups of curcumin have the certain anti-inflammationfunction at 7d, 14d and 28d of medium curcumin group could restrain IPF of rats caused byBLM and medium dose group had the best effect(p＜0.01).The ultra-microstructure observation showed: the lung tissue structure of each group ofcurcumin was integrity to compare with the model group, most of the base membrane wasintegrity, fibroblast hyperplasia and the collagen fiber deposition scope and degree allcompare the model group to ease.Western-blot and immunohistochemistry results show that curcumin can down thecontent of TNF-a in lung tissues of pulmonary fibrosis rats.The primary study of outside factor of lung——circulating fibrocytesUse the model of bleomycin-induced pulmonary fibrosis of rats, immunohistochemicalmethod was used to detect the expression of CD45 and col I in the rat's lungs. The resultsshow that the numbers of circulating fibrocytes which exist in the lungs of bleomycin-inducedpulmonary fibrosis of rats are more then normal control group.2 Experimental studies in vitro2.1 Methods Build up the pulmonary fibrosis model, Adopted the tissue piece primarycultural method to culture the lung fibroblast, stuck wall again and again to turn purely. Withspread generation to be 5-8 generations fibroblast for test object, To investigate the effects ofcurcumin(10, 20, 40, 80and160μmol/L)on proliferation and extracellular matrix secretion ofrat lung fibroblasts in vitro, MTT colorimeter was used for assaying proliferation of LFbs.Typeâ… collage, hyaluronic acid, Laminin and fibronectin were measured by ELISA.2.2 Results The result show: Addition of(10-160)μmol/L curcumin into culture medium hadno cytotoxicity to LFbs, but could significantly inhibit LFbs proliferation, and also inhibitTypeâ… collage, hyaluronic acid, laminin and fibronectin secretion in different degree, with 80μmol/L of curcumin result is the best.Conclusions1 curcumin can level down the content of extracellular matrix and level down the degree ofpulmonary fibrosis in fibrotic rats by bleomycin-induced and thus exertive preventionand the treatment pulmonary fibrosis function.2 curcumin can inhibit the TNF-a activity in the lung epithelioid cell and interstitial cell infibrotic rats by bleomycin-induced, lowering the lung tissue inflammation respond andreducing fiber pilling and restraining the formation of fibrosis.3 The lung fibroblast is the key that the pulmonary fibrosis occurrence and develops,Curcumin can significantly inhibit LFbs proliferation and extracellular matrix secretion,and thus prevent and treat pulmonary fibrosis. 4 Circulating fibrocytes (origins bone marrow) play an important role in bleomycin-inducedpulmonary fibrosis of rats Provide a new way of thinking for the clinic treatment.