| BackgroundTwo types of interstitial cells are widely distributed in the muscularis of the gastrointestinal tract?GI?:interstitial cells of Cajal?ICC?and platelet-derived growth factor receptor?-positive cells?PDGFR?+cells?.ICC play key roles in the generation and propagation of slow waves,and also participate in the neurotransmission in GI.Similar to ICC,PDGFR?+cells are also involved in the neurotransmission.The two types of interstitial cells are adjacent to each other and closely parallel with each other to form a network structure in the muscular layer of GI,which are involved in regulating the contraction and relaxation of smooth muscle.The functional syncytium,consisting of smooth muscle cells?SMC?,ICC and PDGFR?+cells,was named as SMC–ICC–PDGFR?+cell syncytium?SIP syncytium?.And those three kinds of cells were called as SIP cells.SIP cells not only constitute a functional syncytium but also share a close relationship during development.Many studies have revealed that ICC and SMC develop from the same precursor cells during embryogenesis.Blocking c-Kit signaling results in the transdifferentiation of ICC to a smooth muscle phenotype.However,the relationship between PDGFR?+cells and SMC as well as the relationship between PDGFR?+cells and ICC during morphological development remains unknown.C-Kit,a protein-tyrosine kinase receptor,is mainly distributed in ICC in the GI of adult animals and becomes a critical marker molecule of ICC.C-Kit is essential for the development and functional maintenance of ICC.PDGFR?is also an important member of the tyrosine kinase receptor family,which is mainly distributed in PDGFR?+cells as its marker molecule in the GI of adult animals.However,the role of PDGFR?in the development of PDGFR?+cells is still unclear.Research has shown that PDGFR?could positively regulates the c-Kit signaling pathway by stabilizing ETV1 when PDGFR?and c-Kit are coexpressed.A small number of cells coexpress PDGFR?and c-Kit in the GI of adult animals,including 3%-5%of mouse ICC and 35%-44%of ICC precursors.ICC precursor cells are the main source of regenerated ICC and play an important role in maintaining balance of cell number and function stability in ICC networks after birth.However,the role of PDGFR?in the differentiation and maturation of ICC precursors to ICC is still unclear.Moreover,research has shown that PDGFR?and c-Kit are both expressed in the common precursor cells of longitudinal smooth muscle cells?LMC?and ICC during embryonic development,while the selective inhibition of PDGFR suppresses the differentiation of LMC in the embryonic mouse gut.With maturity,the expression of PDGFR?in the gastrointestinal tract is gradually down-regulated and mainly distributed in PDGFR?+cells.Whether the PDGFR?signaling pathway is still involved in the regulation of the development of longitudinal muscle in GI after birth is still unclear.ObjectiveAiming at the above questions,we plan to block the PDGFR?signaling pathway in the gastrointestinal tract of mice by using Crenolanib,a PDGFRs selective blocker,and then observe the morphological changes of SIP syncytium,so as to morphologically explore the role of PDGFR?signaling in the development and phenotypic maintenance of SIP syncytium.At the same time,we further studied the effects of Crenolanib on the expression of functional protein,cell proliferation,apoptosis and function of SIP syncytium,and then explored the role of PDGFR?signaling pathway in the development and functional maintenance of SIP syncytium from the viewpoint of molecular biology,cell behavior and function.Methods1.According to the beginning and ending time of administration of Crenolanib,the experimental group was divided into four groups,including P10 experimental group,P15experimental group,P20 experimental group and P70 experimental group.The mice in P10 experimental group were administered crenolanib from P0 for 10 days.The mice in P15 experimental group were administered crenolanib from P0 for 15 days.The mice in P20 experimental group were administered crenolanib from P10 for 10 days.The mice in P70 experimental group were administered crenolanib from P50 for 20 days.The dose of crenolanib in the P15 intervention group was 7.5 mg?kg·day?-1,while that in the other intervention groups was 5 mg?kg·day?-1.In all intervention groups,crenolanib was administered by gavage.In all control groups,the mice were administered the same volume of normal saline as that of crenolanib used in the intervention group of the same age by gavage.2.Immunofluorescence staining of proximal colonic frozen sections and whole-mount samples of mice in each group was conducted to investigate the effects of Crenolanib on the morphological development,proliferation and apoptosis of SIP syncytium.3.Functional protein expression in SIP cells was detected by Western blotting.4.Colonic transit was analyzed by testing the colonic bead expulsion time to explore the effect of Crenolanib on the function of SIP syncytium.Results1.A dose of 5 mg?kg·day?-1 crenolanib administered for 10 days beginning on P0significantly reduced the number of PDGFR?+cells in the longitudinal muscle?PDGFR?+-LM?and PDGFR?+cells in the myenteric plexus?PDGFR?+-MY?,but it did not affect the PDGFR?+cells in the circular muscle?PDGFR?+-CM?.In addition,crenolanib also caused longitudinal muscle layer thinning without affecting the thickness of the circular muscle layer of colon of P10 experimental group.There was not obvious change in number of ICC in the circular muscularis?ICC-CM?,myenteric plexus?ICC-MY?or longitudinal muscularis?ICC-LM?of colon of P10 experimental group compared with P10 control group.2.When the crenolanib dose was increased to 7.5 mg?kg·day?-1 and the time of administration was extended to 15 days from P0,the number of PDGFR?+-CM,PDGFR?+-LM,PDGFR?+-MY was reduced.And the reduction of PDGFR?+-LM was the most apparent among them.In addition,the number of ICC-CM,ICC-MY,ICC-LM was also reduced.The difference above was statistically significant.3.The number of PDGFR?+-LM was reduced,but the number of PDGFR?+-CM,PDGFR?+-LM and all subtypes of ICC did not change obviously in P20 experimental group compared with P20 control group.Compared with the P70 control,the P70experimental group showed a significant decrease in colonic PDGFR?+-LM,and Crenolanib also caused some PDGFR?+-CM,PDGFR?+-MY,and PDGFR?+-LM to transdifferentiate to an SMC phenotype in P70 experimental group colon.4.Crenolanib reduced the expression levels of functional proteins in smooth muscle cells,ICC,and PDGFR?+cells.Crenolanib did not affect the proliferation and apoptosis activity of PDGFR?+cell.Crenolanib also caused a significant increase in colonic bead expulsion time,which meant a delayed colonic transit.ConclusionsCrenolanib can inhibit the development of SIP cells,especially PDGFR?+cells,which suggests that the development of SIP cells depend on the PDGFR?signaling,and the dependence of PDGFR?+cells are the highest among SIP cells.In addition,different subtypes of PDGFR?+cell have different sensitivity to Crenolanib,and PDGFR?+-LM has the highest sensitivity,which indicates that there is a subtype difference between different subtypes of PDGFR?+cell in the dependence on PDGFR?signaling.With maturity,the sensitivity of PDGFR?+cells to Crenolanib decreases,which means that the dependence of PDGFR?+cells on PDGFR?signaling decreased with maturity.However,the PDGFR?signaling is still involved in the phenotypic maintenance of PDGFR?+cells in adulthood.And blocking PDGFR?causes PDGFR?+cell to transdifferentiate to an SMC phenotype,indicating that PDGFR?+cells originate from smooth muscle cell precursor cells as well as ICC.That is,SIP cells share common progenitors in the GI.Crenolanib resulted in a delayed colonic transit,indicating that blocking PDGFR?signaling can affect the function of SIP syncytium and cause abnormal colonic motility.Down-regulation of the expression of functional protein in SIP syncytium may be the molecular basis for the abnormal function of SIP syncytium. |
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