ABSTRACT
Aim To study the effeet of Wenyang Hua- Nrpl signaling pathway in systemic sclerosis ( SSc ) zhuo Tongluo formula ( WYHZTLF) on the Sema3A/ mouse model, and to explore its mechanism in the treatment of SSe.Methods The systemic sclerosis mouse model was constructed and divided into control group, model group, WYHZTLF low (21 g • kg 1 ) , medium (42 g • kg 1 ) , high (84 g • kg 1 ) dose group and the Sema3A inhibitor epigallocatechol gallate (EGCG) group.All groups were given intragastric administration for four weeks, and the control and model groups were treated with saline.Histopathology and dermal thickness were detected by HE, VEGFA pro-tein expression levels were detected by immunohisto- chemistry; the protein expression levels of Sema3A, Nrpl and VEGFA were determined by Western blot; Sema3A expression levels in mouse serum were detec-terl by ELISA.Results Comparer] with model group, WYHZTLF significantly alleviated the skin lesions in systemic sclerosis mouse model, significantly inhibited the dermal thickness, inhibited the protein expression levels of VEGFA, Sema3A and Nrpl , and reduced the expression levels of serum Sema3A.Conclusion WYHZTLF can improve the vascular injury of SSc, and its mechanism may be related to the inhibition of VEGFA and Sema3A/Nrpl signaling pathway.
ABSTRACT
Nonspecific low back pain is closely associated with afferent nerve ingrowth into degenerated IVDs and increasing the inflammatory response. Members of the class 3 semaphorins signal their response through two prominent receptors; the NRP (Neuropilin-1) and the Plexin A. Sema3A (Semaphorin3A) is primarily known for their role in modulating neuronal survival as well as neurite outgrowth and guidance via regulation of Sema3A-NRP-1-plexinA signal pathway. Also, sema3A is shown to be conductive to innervate the inner painful degenerated IVDs (Intervertebral discs). Furthermore, sema3A is thought to act as a barrier to endothelial cells survival and migration on vascular endothelial growth factor (VEGF) and inhibition of KLF5-induced (Krüppel-like factor 5) inflammatory mediators within degenerated IVDs. Therefore, Sema3A produce a new perspective of dual-action therapeutic agent for attenuating the regulator of innervation and angiogenesis into degenerated IVDs and inhibition of KLF5-induced inflammation.
Subject(s)
Humans , Endothelial Cells , Low Back Pain , Neuropilin-1 , Semaphorin-3A , Vascular Endothelial Growth Factor AABSTRACT
Objective To investigate the effect of Semaphorin3A (Sema3A) on axon growth of primary retinal ganglion cells (RGCs) in mice.Methods C57/BL6 mice within 24 hours after birth were sacrificed and the eyeballs were removed,RGCs was isolated from retina and cultured in vitro.The primary cultured RGCs was identified by Brn3a immunofluorescence staining.Seven days after culture,the RGCs was divided into control group,0.05 μg/ml Sema3A group,0.10 μg/ml Sema3A group and 0.50 μg/ml Sema3A group,and the processing time was 2 hours.Immunofluorescence staining was used to detect the expression of neuron-specific marker β3-tubulin and dendritic marker MAP2,β3-tubulin+/MAP2-was identified as axons.The length of axons was measured by Image J software.The axon lengths of 0.10 μg/ml Sema3A group and control group at 0.5,1 and 2 hours after treatment were calculated.The primary cultured cells were divided into control group,Sema3A treatment group,Y27632 treatment group and combined treatment group according to different drugs.The average axon lengths were compared among the groups.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO).Results Brn3a was positively expressed in primary cultured RGCs as a specific cell marker.Seven days after culture,RGCs tended to mature,with complete elongated neuronal processes and branches.The axon lengths of 0.05,0.10 and 0.50 μg/ml Sema3A groups were (69.35±1.49),(60.45±1.42) and (93.65±1.86) μm,which were significantly shorter than (109.80±2.29) μm of the control group (all at P＜0.01).The axon length of 0.10 μg/ml Sema3A group was (165.00 ±4.39)μm and (97.63 ±2.79)μm at 1 hour and 2 hours after treatment,respectively,which was significantly lower than (210.40 ±4.44) μm and (199.00 ± 4.36) μm of control group at corresponding time points (both at P＜0.01).There was a significant difference in the axon length of control group,Sema3A treatment group,mixed treatment group and Y27632 treatment group (F =142.50,P＜0.01).The RGC axon length of Sema3A treatment group was significantly lower than that of control group and combined treatment group (both at P＜0.01).Conclusions Sema3A can inhibit axon growth of primary retinal RGCs in mice,and ROCK signaling pathway inhibitors can alleviate such restrain effect.
ABSTRACT
Objective:To explore the role of Semaphorins 3A(Sema 3A) and its receptor Neuropilin-1 (Nrp1) in the development of chronic periodontitis of rats and clinical samples.Methods:20 SD rats were divided into 2 groups.Rats in the experimental group were induced into chronic periodontitis models.Rats in control group were not treated.After 8 weeks,maxilla of all the rats were collected for micro-CT scanning and IHC staining.The distance from cementoenamel junction to alveolar bone crest(CEJ-ABC) and the IOD of Sema3A/Nrp1 positive staining in rats were analyzed.20 clinical samples of chronic periodontitis(n =10) and normal periodontal tissues (n =10) were collected for immunofluorescence and qRT-PCR analysis.Results:The CEJ-ABC distance of chronic periodontitis group was higher than that of the control group(P ＜ 0.05).The IOD of Sema3A/Nrp1 in experimental group was lower than that of the control group (P ＜ 0.05) and with a negative correlation with bone loss (P ＜ 0.05).Immunofluorescence and qRT-PCR analysis showed that the expression level of Sema3a/Nrpl in clinical samples with chronic periodontitis was also lower than that of the healthy subjects(P ＜ 0.05).Conclusion:The reduced Sema3A/Nrp1 plays an important role in the development of bone destruction in chronic periodontitis.
ABSTRACT
Objective To construct and identify over?expressing lentiviral vector of mSema3A. Methods Sema3A gene of mice was amplified by PCR,then the gene was inserted into plasmids pDown?mSema3A?IRES/EGFPby Gateway technology. The plasmids pLV/EXPNZ?puro?mSema3A?IRES/EGFP were produced by recombination. After sequencing identification,the vector pLV(Exp)?Puro?CMV?mSema3A?IRES/EGFP was packed and condensed. Finally the recombinant vectors were used to transfect 293T cells to obtain virus pools. Results The recombinant lentiviral vectors were 11 538 bp with EGFP marker,and Sema3Agenes were inserted into the lentiviral vector correctly,indicating the over?expressing vector of Sema3A gene in mice was successfully constructed. Conclusion The over?expressing lentiviral vector of mSema3A was constructed correctly, which lay a foundation of screening of over?expressing strains of such gene in specific cells.
ABSTRACT
Neuropilin 1 (NP1) is a part of essential receptor complexes mediating both semaphorin3A (SEMA3A) and vascular endothelial growth factor (VEGF) which is one of important mediators involved in the pathogenesis of asthma. Therefore, it is possible that SEMA3A plays a role in the pathogenesis of asthma through attenuation of VEGF-mediated effects. In the present study, we aimed to evaluate expression levels of SEMA3A and NP1 using induced sputum of asthmatics and a murine model of asthma. Firstly, SEMA3A and NP1 expressions in induced sputum of asthmatics and SEMA3A and NP1 expression on bronchoalveolar lavage (BAL) cells and lung homogenates of asthmatic mice were determined. Then we evaluated the immunolocalization of VEGF receptor 1 (VEGFR1), VEGF receptor 2 (VEGFR2), and NP1 expressions on asthmatic mice lung tissue and their subcellular distributions using fibroblast and BEAS2B cell lines. Sputum SEMA3A and NP1 expressions were significantly higher in asthmatics than controls. Similarly, SEMA3A and NP1 expressions on BAL cells and lung homogenates were significantly elevated in asthmatic mice compared to control mice. Immunohistochemical analysis showed that VEGFR1, VEGFR2, and NP1 expressions were also uniformly increased in asthmatic mice. Our observations suggest that SEMA3A and NP1 may play important roles in the pathogenesis of asthma.