The Effect Of Isopsoralen On Congenital Craniovertebral Malformations And TGF-β/Smads Signaling Pathway

The craniovertebral junction（CVJ）is composed of the foramen magnum region,Atlas,axis,the second and third intervertebral disc as well as the surrounding soft tissue,which are adjacent to the medulla and is the main motor unit of head and neck.Congenital malformations of CVJ are mainly due to abnormal gene expression during embryonic development period,which most commonly involve the vertebrobasilar artery and medulla and seriously affect the movement of the neck,or even cause paralysis and death.For now,however,there are lack of researches on the pathogenesis and there are no effective control measures for this disease.In consequence,patients with serious compression symptoms of spinal cord and nerve have to receive surgical treatments.However,current surgical procedures still have high difficulties and risks.So CVJ was often referred to as the "danger zone" and "forbidden zone" in spinal surgery.Isopsoralen is an effective component of Psoralea corylifolia for the treatment of some orthopedic diseases,which can improve proliferation and differentiation of osteoblast and the mechanism is closely related to the TGF-β/Smads signaling pathway.The present research is mainly aim to investigate the effect of isopsoralen on congenital craniovertebral malformations on animal and cell level and preliminarily explore the correlation between TGF-β 1/smad signaling pathway and Hox gene expression in the development of CVJ.Part Ⅰ Effect of decreased expression of Hoxd3 gene on craniovertebral development and the correlation to vitamin A deficiencyObjective:In this experiment,the rat model of congenital craniovertebral malformations was established by intervention of vitamin A deficiency（VAD）that was aim to observe the malformations of the CVJ,and invistigate the formation mechanism of congenital craniovertebral malformations caused by decreased expression of Hoxd3 gene as well as the correlation to VAD.Methods:30 weaning SD female rats were randomly divided into normal control group（N）and VAD group.Rats in VAD group was fed with VA deficient diet,and rats in N group was fed with normal diet.When female rats in group VAD had significant VA deficiency symptoms and VA level in serum were significantly lower than 200 ng/ml,the two groups of rats were mated with male rats.half of embryos in each group were used to detect the mRNA expression of Hoxd3 and retinoic acid receptors（RARs）by RT-qPCR during the day 11.5 of embryonic（E11.5）;On the E21,whole skeletal specimens of neonatal rats disposed by compound staining method,and the bony structure of CVJ were observed by stereoscope.Results:The mRNA expression of Hoxd3 and RARs were significantly decreased in VAD group（P<0.05）;Bony structure of CVJ in neonatal rats were found obviously malfomation by stereoscope,such as atlas and axis dysplasia,bony fusion or turbulence of atlantooccipital and atlantoaxial joint.Conclusions:The results showed that the decrease mRNA expression of Hoxd3 gene can cause the development malformations of CVJ.Besides,we also found that the expression of RARs was also significantly decreased,which suggested that RA plays a role in development of CVJ through binding with its receptors.Part Ⅱ Effect of isopsoralen on TGF-β1/smad signaling pathway in correcting congenital craniovertebral malformationsObjective:In this part of experiment,rats of medication groups were treated with isopsoralen.The expression of Hoxd3 and TGF-β1 genes and related Smads were detected to investigate the effect on congenital craniovertebral malformations and preliminarily explore the correlation between TGF-β1/smad signaling pathway and Hox gene expression in the development of CVJ.Methods:60 weaning SD female rats were randomly devided into normal control group（N）,model group（VAD）,different doses（low,medium and high）of isopsoralen intervention group（ISL,ISM,ISH）.Modeling method of group VAD,ISL,ISM,ISH was same with the Part 1.On the E0.5,the rats in medication groups were treated with continuous intragastric administration of isopsoralen till to E21.RT-qPCR was used to detect the mRNA expression of Hoxd3,TGF-β1 Smad4,Smad7 gene in the embrys on E11.5;Immunohistochemical-staining was used to detect the protein expression of TGF-β1 in the CVJ of neonatal rats,and skelekton structure of CVJ was observed by stereoscope.Results:（1）Typical craniovertebral malformations were found in neonatal rats in VAD group;On the other hand,there was lower incidence of craniovertebral malformations in ISL,ISM,ISH groups and dgree of malformations were improved which compared to VAD group.But compared with the N group,neonatal rats in these groups still had malformations and malformations rate between these groups had no significant difference;（2）In ISL,LSM,LSH group,these were significantly increased in mRNA expression of Hoxd3 gene compared to the VAD group（P<0.05）.However,along with the increasing dosage of isopsoralen,there were no significantly difference in mRNA expression amog these groups.And mRNA expression of TGF-β1 and Smad4 gene in these groups were significantly increased compared to VAD group（P<0.05）,while on the contrary mRNA expression of Smad7 was inhibited in these groups（P<0.05）;（3）There were significantly difference in protein expression of TGF-β1 between ISL,LSM,LSH group and VAD group（P<0.05）as well as the expression was significantly decreased in VAD group compared to N group（P<0.05）.Conclusion:（1）The reults suggested that these dose of isopsoralen can reduce the incidence of congenital craniovertebral malformations and improve the degree of malformations.We conclude that isopsoralen can regulate expression of Hoxd3 gene by increasing the TGF-(31/smad signaling pathway.（2）There are interaction between RA/RARs signaling pathway and TGF-β1,as VA deficiency could cause decrease of TGF-β1 expression in the CVJ of the neonatal rats on the transcription and translation levels.Part Ⅲ Effects of isopsoralen on BMP-2/Smad signaling pathway in osteroblast cell Objective:This part of the experiment is aim to observe the proliferative effects of isopsoralen on MC3T3-E1 cell and to investigate the correlation between effect of isopsoralen on promoting bone formation and BMP-2/Smad signaling pathway by detecting mRNA and protein expression of BMP-2,Smadl and Smad5.Methods:MC3T3-El cells were cultured in vitro.Different concentrations of isopsoralen(10^-1,10^-2,10^-3-10^-4M)were incubated with 72 h comparing with the 0 M as control.MTT method was used to detect the effect of isopsoralen on cell proliferation activity;The mRNA and protein expression of BMP-2,Smadl and Smad5 were detected by RT-qPCR and Western-blot respectively.Results:10^-1,10^-2,10^-3,10^-4M isopsoralen promoted proliferation of MC3T3E-lcell in concentration-dependent manners;These concentrations of isopsoralen significantly incresased mRNA and protein levels of BMP-2,Smadl,Smad5（P<0.05）,of which 10^-2 M isopsoralen had strongest stimulating effect,and 10^-1 M was weaker slightly.But there were no significant differences between the two concentration（P>0.05）.Conclusion:Isopsoralen obviously promote the proliferation of MCT3T3-E1 cells cultured in vitro;The promotion of isopsoralen was related to BMP-2/smad pathway.