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OBJECTIVE@#To investigate the effects of inhibiting Sonic Hedgehog (Shh) signaling on fibrous scar formation and functional outcome after ischemic brain injury.@*METHODS@#Adult SD rats were randomized into sham-operated group, middle cerebral artery occlusion (MCAO) and reperfusion (I/R) group, I/R with intraventricular empty adenoviral vector (rAd-NC) injection group, and I/R with adenovirus-mediated Shh knockdown (rAd-ShShh) group. After the treatments, the neurological deficits of the rats were assessed, and the protein and mRNA expressions of fibronectin (Fn), α-SMA, and Shh in the ischemic hemisphere were detected with immunofluorescence assay and qPCR; TUNEL staining was used for detecting neural cell apoptosis. In the cell experiment, primary meningeal fibroblasts isolated from neonatal SD rats were pretreated for 24 h with TGF-β1 or TGF-β1 plus cyclopamine (CYC) before oxygen-glucose deprivation for 150 min followed by reoxygenation for 72 h (OGD/R). CCK-8 assay and scratch test were performed to examine the changes in cell proliferation and migration, and immunofluorescence assay, qPCR and Western blotting were used for detecting cell transformation and the expressions of Shh, α-SMA, and Fn.@*RESULTS@#Cerebral I/R injury significantly increased the protein and mRNA expressions of Shh, α-SMA, and Fn in the ischemic hemisphere of the rats, but their expression levels were significantly lowered by intraventricular injection of rAd-Shshh (P < 0.05), which obviously increased cell apoptosis in the ischemic hemisphere (P < 0.05) and improved modified mNSS and modified Bederson scores of the rats (P < 0.05). In the cell experiment, pretreatment with TGF-β1 and TGF-β1+CYC both increased the viability of the primary meningeal fibroblasts after OGD/R. TGF-β1 significantly enhanced the migration ability and induced obvious transformation of the exposed cells (P < 0.05), but these effects were significantly attenuated by co-treatment with CYC (P < 0.05). The expressions of Shh, α-SMA and Fn in the TGF-β1 group were all significantly higher in TGF-β1-treated cells (P < 0.05) and were obviously lowered by co-treatment with CYC (P < 0.05).@*CONCLUSION@#Inhibition of Shh signaling may inhibit fibrous scar formation and functional recovery in rats after ischemic brain injury.
Subject(s)
Animals , Rats , Brain Injuries , Cicatrix , Hedgehog Proteins , RNA, Messenger , Rats, Sprague-Dawley , Transforming Growth Factor beta1ABSTRACT
Aim To explore the effect of miR-152 on proliferation of cardiac fibroblasts ( CFS) in diabetic cardiomyopathy. Methods Diabetic cardiomyopathy model was established in SD rats by STZ injection, and CFS proliferation model was established by high glucose (33. 3 mmol • L ~1). HE and Masson staining were performed in paraformaldehyde fixed myocardium of rats. Western blot determined a-SMA and collagen I protein expression. qPCK detected gene expression of miR-152. MTT assay analyzed the proliferation of cells. Results HE and Masson staining showed the higher level of myocardial collagen in diabetic cardiomyopathy model. Furthermore, the myocardial myo-cytes lined up in disorder. Western blot showed that the expressions of a-SMA and collagen I were up-regulated in the diabetes mellitus ( DM ) group, while the expression of miR-152 was down-regulated. The result of the in vitro experiment showed that a-SMA and collagen I expressions were down-regulated after trans-fected miR-152 mimics. The proliferation of CFS was also down-regulated after transfected miR-152 mimics. Conclusions miR-152 plays an important role in the proliferation of CFS and may ameliorate diabetic cardiomyopathy.
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Aim To explore the inhibitory effects of resveratrol (Res ) on human pulmonary fibroblast growth,and its related mechanisms.Methods Hu-man pulmonary fibroblasts MRC-5 were cultured in vitro as research object.These cells were inoculated with 20 μL dimethyl sulfoxide (DMSO)as well as 0, 12.5,25 50,100 and 200 μmol·L-1 Res for 24,48 and 72 h,respectively.Inhibitory rate of cellular pro-liferation was analyzed by MTT.In addition,these cells were treated with 20 μL DMSO (medium group) as well as 50 and 100 μmol·L-1 Res for 48 h,re-spectively.Subsequently,cell cycle and apoptotic rate were measured using flow cytometry.Apoptosis index (AI ) was detected through terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The mRNA and protein expression of cell cycle protein D1 (Cyclin D1 ) and cyclin-dependent kinase 4 (CDK4)was detected through fluorescence real-time quantitative PCR and Western blot, respectively. Western blot was used to measure the protein expres-sion of Bcl-2 and Bax.Results With the increase of Res concentrations and prolongation of treated time, inhibitory rate of cellular proliferation was gradually el-evated (P<0.01).After 48 h of co-culture,DNA ra-tio of S and G2/M periods,mRNA and protein levels of Cyclin D1 and CDK4,and Bcl-2 protein levels were significantly decreased while DNA ratio of G0/G1 peri-od,AI,apoptotic rate and Bax protein levels were sig-nificantly increased in 50 and 100 μmol · L-1 Res-treated groups as compared to medium group (P <0.01 ).Moreover,the effects 100 μmol · L-1 Res were better than those of 50 μmol · L-1 Res (P <0.01).Conclusion Res can suppress the prolifera-tion of MRC-5 cells,which may be associated with blockade of cell cycle progression and induction of cell apoptosis.
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Objective To examine the expression of p44/42 mitogen -activated protein kinase (p44/42MAPK) and Phospho-p44/42MAPK in cicatrix (hy pertropic scars and keloids) and to aim at exploring the role of activated p44/ 42 MAPK pathway in development of cicatrix. Methods In or der to analyse the differences, 10 samples of normal skin (NS), hypertropic scar s (HS) and keloids (K) were collected, and then the extracted cytoplasmic protei ns from each tissue were examined by Western blotting for p44/42 MAPK and Phosph o p44/42MAPK and immunohistochemical staining with specific antibodies was emplo yed to determine that in fibroblasts of K, HS and NS. Results There was no evident difference of p44/42MAPK in the tissues and fibroblas ts between cicatrix and NS, but Phospho-p44/42MAPK was obviously higher in cica trix than that in NS. In cicatrix, there was no evident difference of p44/42MAPK in tissues between HS and K, while in fibroblasts, Phospho-p44/42MAPK in K was much higher than in HS. Conclusion Activation of p44/42MA PK pathways involves in formation of cicatrix.