Pirfenidone suppresses the abnormal activation of human Müller cells after platelet-derived growth factor-BB stimulation

AIM: To determine the effect of pirfenidone on the activated human Müller cells by platelet-derived growth factor-BB(PDGF-BB). METHODS: The primary human Müller cells were separated from retinal tissues and established the pathogenic model by stimulated with PDGF-BB. The Müller cells behaviour of normal group and the model group was measured by MTT assay, Trypan blue assay, cell migration assay, and collagen contraction assay. The expression of transforming growth factor(TGF)-β1,-β2, and pigment epithelium-derived factor(PEDF) was estimated with realtime polymerase chain reaction(PCR), Western blot and immunofluorescence analyses. RESULTS: A pathogenic/proliferative model of Müller cells was established by stimulating normal cultured Müller cells with 10 ng/mL PDGF-BB for 48 h. After treated with 0.2 and 0.3 mg/mL pirfenidone, the proliferation, migration and collagen contraction was statistically significantly depressed in the model group compared with the normal groups. The expression levels of TGF-β1 and TGF-β2 were significantly down-regulated, while the PEDF expression was significantly up-regulated after treated with 0.2 and 0.3 mg/mL pirfenidone in the model group. CONCLUSION: Pirfenidone effectively suppress the proliferation, migration and collagen contraction of the human Müller cells stimulated with PDGF-BB through down-regulation of TGF-β1/TGF-β2 and up-regulation of PEDF.

Distribution of platelet-derived growth factor-alpha receptor expressing oligodendrocyte precursor cells in the adult rat brain

BACKGROUND： Studies have demonstrated that NG2-positive glial cells in the adult rats are predominantly located in the gray and white matter of the cerebral cortex and hippocampus. Platelet-derived growth factor-a receptor （PDGF-αR） cells are a subset of oligodendrocytes, which are not as mature as NG2-positive cells. Distribution and migration of PDGF-αR-positive cells in the rat brain remain poorly understood. OBJECTIVE： Using immunohistochemical methods, the distribution of oligodendrocyte precursor cells （PDGF-αR-positive） was analyzed in the adult rat brain. DESIGN, TIME AND SETTING： Immunohistochemical study was performed at the Department of Histology and Embryology of the Third Military Medical University from September 2007 to September 2008. MATERIALS： Rabbit anti-PDGF-αR polyclonal antibody was purchased from Santa Cruz Biotechnology, USA. Streptomycin-avidin-biotin complex immunohistochemistry kit was purchased from Zhongshan Goldenbridge Biotechnology, China. METHODS： Whole brains from 5 healthy, adult, Wistar rats were collected for immunohistochemistry, and the mean value of PDGF-αR-expressing cells was quantified. The absolute values were translated to ranked data of high, moderate, and low grades （high grade： 10 positive cells; moderate grade： 5-9 cells; low grade： 〈 5 cells in a 400 × visual field）. Based on the number of cell processes and branches, as well as the number of PDGF-αR-positive cells, in different regions, the cells were classified into three categories, i.e., type Ⅰ-Ⅲ. From type I to type Ill, the number of processes gradually increased. MAIN OUTCOME MEARSURES： The number and distribution of PDGF-αR-positive cells in different brain regions of adult rats. RESULTS： PDGF-αR-positive cells were located in the forebrain and midbrain, but not in the cerebellum or brainstem. In the olfactory bulb and hippocampus, a total of 60% PDGF-αR-positive cells were type Ⅰ and these cells were not mature as others. In the cerebral cortex, olfactory system, hippocampus, and optic chiasma, where neuronal bodies aggregated, approximately 40% of the PDGF-αR-positive cells were type Ⅱ, with few type Ⅲ cells. In the white matter, corpus callosum, basal nucleus, and thalamus, PDGF-αR-positive cell density was moderate. In the olfactory bulb and hippocampus, PDGF-αR-positive cell density was high. PDGF-αR-positive cells were not observed in the cerebellum or brainstem CONCLUSION： PDGF-αR-positive cells were aggregated in the olfactory bulb and hippocampus in the adult, rat brain, but few cells were detected in the cerebellum and brainstem.

Effect of IL-10 on the expression of HSC growth factors in hepatic fibrosis rat

AIM： To study the effect of IL-10 on the expression of growth factors - transforming growth factor-β1 （TGF-β1）, epidermal growth factor （EGF）, hepatocyte growth factor （HGF）and platelet-derived growth factor （PDGF） of hepatic stellate cells （HSCs） of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10. METHODS： Hepatic fibrosis was induced by CCI4 administration intra-peritoneally. Sixty clean male Sprague-Dawley （SD） rats were randomly divided into three groups： normal control group （GN, 8 rats）, hepatic fibrosis model group （GC, 28 rats） and IL-10 treated group （GI, 24 rats）. At the beginning of the 7^th and 11^th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs. RESULTS： Rat hepatic fibrosis was developed with the increase of injection frequency of CCl4, and HSCs were successfully isolated. At the 7^th and 11^th wk, TGF-β1, EGF, and HGF mRNA in GC increased obviously compared with GN （P = 0.001/0.042, 0.001/0.001, 0.001/0.001） and GI （P= 0.001/0.007, 0.002/0.001, 0.001/0.001）. For TGF-β1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7^th wk （P= 0.005） and 11^th wk （P= 0.049）. For HGF, mRNA level in GI decreased compared with GN at the 7^th wk （P = 0.001） and 11^th wk （P= 0.021）. Between these two time points, TGF-β1 expression at the 7^th wk was higher than that of the 11^th wk （P = 0.049）, but for EGF, the former was lower than the latter （P = 0.022）. As for PDGF mRNA, there was no significant difference between thesegroups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-β1 and EGF were paralleled with the above findings. CONCLUSION： The expression of TGF-β1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.