ABSTRACT
Objective To investigate the effects of pirfenidone on orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO) and its underlying mechanisms.Methods OFs from patients with TAO were isolated and cultured in DMEM.Cells were divided into four groups and treated with 0,250,500 and 1 000 μg/ml pirfenidone for 24,48 or 72 hours,respectively.Cell proliferation was detected by tetramethyl azo salt (MTT) assay,and cell viability was determined by trypan blue.Transforming growth factor (TGF) β1 mRNA level was determined by real-time fluorescence quantitative PCR (RT-qPCR).Type Ⅰ and type 11Ⅲ collagen secreted from cultured cells were measured by enzyme-linked immuno sorbent assay (ELISA).Results (1) The primary cultured OFs had typical fibroblast spindle-like morphology.(2) MTT assay showed that pirfenidone treatment significantly inhibited the proliferation of OFs in a dose-dependent manner (P＜0.05) with the proliferation rates of pirfenidone treated groups of-15.31％,-24.92％,-48.53％ from 250,500,1 000 μg/ml after 72 h,respectively,in which the inhibition effect of 1 000 μg/ml pirfenidone was significantly different from the other two treated groups (P＜0.05).There were no significant differences in the inhibitory effect of the same concentration group among different time points at 24 h,48 h and 72 h (P＞0.05).Trypan blue showed that the survival rate of OFs in different concentrations of pirfenidone from 0,250,500,1 000 μ-g/ml at 72 h were 78.37％,79.21％,78.24％ and 76.28％,respectively.There were no significant differences between each drug treated and the control group (P＞0.05).(3) RT-qPCR results showed that the mRNA expression levels of TGFβ1 at 250,500,1 000 μg/ml pirfenidone treated groups at 72 h were 0.760±0.010,0.440±0.006,and 0.290±0.002,respectively.Compared with the control group (0.950±0.014),the differences were statistically significant (all P＜0.05).Moreover,TGFβ1 mRNA expression level in 1 000 μg/ml pirfenidone treated group was significantly lower than those in the other two treated groups (all P＜0.05).The secretion of type Ⅰ collagen (0.633 ± 0.006,0.527 ± 0.003 and 0.402±0.008) and type 11Ⅲ collagen (0.511±0.003,0.439±0.007 and 0.223±0.006) in 250,500 and 1 000 μg/ml pirfenidone treated groups at 72 h were significantly lower than those in the control group (0.794±0.005,0.527±0.007,all P＜0.05).Type Ⅰ and type Ⅲ collagen secretion in 1 000 μg/ml pirfenidone treated group were significantly lower than those in the other two groups (P＜0.05).Conclusions Pirfenidone inhibits the cell proliferation,TGFβ1 expression and collagen secretion of OFs,which may contribute to the anti-fibrotic effect of pirfenidone.
ABSTRACT
Objective To explore the mechanism of bone marrow-derived endothelial progenitor cells (EPCs)in the treatment of peripheral neuropathy of diabetic rats. Methods Rats with diabetic peripheral neuropathy (DPN)were induced by streptozotocin(60 mg/kg). Male SD rats were divided into normal control group(NC group),DPN group, DPN+saline group(DPN+S group), and DPN+ EPCs group. Sciatic nerve motor nerve conduction velocity(MNCV)was measured. The expressions of NF-κB and myelin basic protein(MBP)in sciatic nerve were detected by immunohistochemistry. Results Compared with DPN group,the expression of NF-κB was reduced in the sciatic nerve of DPN+EPCs group,while the expression of NF-κB was increased in the sciatic nerve of DPN+ S group. There was no statistical difference in the expression of MBP between DPN and DPN+ EPCs groups. Compared with DPN+S group, the expression of MBP was higher in DPN+EPCs group. Conclusion Transplantation of EPCs inhibits the expression of NF-κB and increases the expression of MBP, which might be conducive to the repairs of nerve injury.
ABSTRACT
Objective To explore whether endothelial progenitor cells (EPCs) exerts therapeutic effects on rats with diabetic peripheral neuropathy (DPN).Methods Diabetes was induced by streptozotocin.Male SD rats were divided into normal control group(NC group), diabetic peripheral neuropathy group(DPN group), DPN+saline group(DPN+S group), and DPN+EPCs group.Sciatic nerve motor nerve conduction velocity(MNCV) was measured by a electromyography and trigger potentiometer instrument.Pathological changes were observed under optical microscope and electron microscope.Results Compared with normal control group [(52.91 ± 4.53) m/s], both DPN group and DPN+S group had slower sciatic nerve MNCV [(36.51 ± 6.30 and 25.37 ± 5.48) m/s, P＜0.01].Compared with DPN group, the MNCV of sciatic nerve in DPN + EPCs group had improved significantly [(36.51 ± 6.30 vs 46.20 ± 11.70) m/s, P ＜ 0.01].Sciatic nerve pathological changes, characterized by demyelination and axonal degeneration under light microscope and electron microscope were observed in DPN group, DPN+EPCs group, and DPN+S group.Compared with DPN+S group, the sciatic nerve pathological changes of DPN+EPCs group were alleviated.Conclusion After fed for 12 weeks, diabetic rats could progress to DPN rats.Transplantation of EPCs could improve MNCV, demyelination and axonal degeneration changes of sciatic nerver of the DPN rats.