ABSTRACT
Objective To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on autophagy and the secretion of chemokine receptor CXCR4 induced by low-dose immunosuppressive durgs.Methods Flow cytometry was used to detect the changes of hUC-MSCs surface markers after treatment with low-dose tacrolimus and rapamycin.The effect of treatment with tacrolimus and rapamycin on proliferation of hUC-MSCs was analyzed with WST-1 assay.Regular RT-PCR was applied to analyze the mRNAs expression of ligands such as LC3B,Atg5 and Beclin1 in hUC-MSCs.Western blotting was carried out to detect the expression of LC3B,Atg5,Beclin1 and p-ULK1 in hUC-MSCs after treatment with tacrolimus and rapamycin.The secretion of chemokine receptor CXCR4 in hUC-MSCs was analyzed under the state of autophay by flow cytometry.Results Flow cytometry analysis confirmed low-dose immunosuppressive drugs tacrolimus and rapamycin did not cause changes in hUC-MSCs phenotypes significantly.Low-dose tacrolimus had no cytotoxic effect on hUC-MSCs,while,rapamycin could inhibit the proliferation of hUC-MSCs after 24 h or 48 h,with survival rate being 73.66％ and 68.81％ (P＜0.05) of controls,respectively.Moreover,both tacrolimus and rapamycin could inhibit PI3K/AKt/mTOR signaling pathway to activate hUC-MSCs autophagy,and the related proteins of LC3B,Atg5 and Beclin1 increased significantly and induced the up-regulation of CXCR4 secretion.Conclusion Our results here demonstrated that low-dose tacrolimus and rapamycin induce autophagy in hUC-MSCs and promote the secretion of CXCR4.
ABSTRACT
Neurons in the laterodorsal tegmentum (LDTg) and pedunculopontine tegmental nucleus (PPTg) play important roles in central autonomic circuits of the kidney. In this study, we used a combination of retrograde tracers pseudorabies virus (PRV)-614 and fluorescence immunohistochemistry to characterize the neuroanatomic substrate of PPTg and LDTg innervating the kidney in the mouse. PRV-614-infected neurons were retrogradely labeled in the rostral and middle parts of LDTg, and the middle and caudal parts of PPTg after tracer injection in the kidney. PRV-614/TPH double-labeled neurons were mainly localized in the rostral of LDTg, whereas PRV-614/TH neurons were scattered within the three parts of LDTg. PRV-614/TPH and PRV-614/TH neurons were located predominantly in the caudal of PPTg (cPPTg). These data provided direct neuroanatomical foundation for the identification of serotonergic and catecholaminergic projections from the mid-brain tegmentum to the kidney.