| Objective:Promoting angiogenesis is of great significance to tissue repair.A large number of studies have shown that adipose-derived mesenchymal stem cells(ADSCs)can release a variety of pro-angiogenic cytokines,and can also differentiate endothelial-like cells to directly participate in angiogenesis.It is an important vascularizing seed cell in regenerative medicine and tissue engineering.Transient receptor potential vanilloid 1(TRPV1)is an important target for the treatment of ischemic diseases.It exists in endothelial cells and smooth muscle cells,and participates in the regulation of angiogenesis.Our previous study found that TRPV1 was also expressed in rat ADSCs,and activation of TRPV1 could promote the proliferation of ADSCs.Therefore,this study intends to explore the effect of the TRPV1 agonist Capsaicin(Cap)on the vascularization of ADSCs from the aspects of endothelial differentiation and paracrine capacity of ADSCs,and to determine whether Cap pretreatment enhances the promotion of ADSCs vascularization at the global and ex vivo levels.Methods:(1)Rat primary ADSCs were extracted by enzymatic digestion,and the surface markers of ADSCs were identified by flow cytometry: positive markers CD29 and CD90,and negative markers CD31 and CD45.(2)The effects of Cap and TRPV1 antagonist Capsazepine(CAPZ)on the induction of ADSCs into endothelium were explored: In the process of endothelial differentiation,set the group control,Cap(1,3,10 μM),CAPZ(1,3,10 μM).The changes of cell morphology were observed under a microscope,and the expression of vascular endothelial marker protein CD31 in ADSCs was detected by Western blotting.(3)The effects of Cap and CAPZ on the paracrine secretion of vascular endothelial growth factor(VEGF),fibroblast growth factor(b FGF)and nitric oxide(NO)in ADSCs under normal glucose(5.5 m M)and high glucose(33 m M)environments were explored.Normal glucose: ADSCs were incubated in normal glucose medium with different concentrations of Cap(0,1,3,10 μM)and CAPZ(1,3,10 μM)in 2% FBS for 48 h,and the cell culture supernatant was collected.High glucose: ADSCs were incubated in high glucose medium of 2% FBS with different concentrations of Cap(0,1,3,10 μM),and the medium was changed every 48 h to add drugs for a total of 4 days,and the cell culture supernatant was collected.The supernatant was centrifuged to obtain ADSCs conditioned supernatant(ADSCs-CM).The levels of VEGF and b FGF in ADSCs-CM were detected by ELISA kit,and the level of NO in ADSCs-CM was determined by Griess reagent system.(4)The effects of Cap pretreatment on ADSCs-CM in promoting tube-like formation in human umbilical vein endothelial cells(HUVECs)in vitro were explored:HUVECs were placed on Matrigel,and ADSCs-CM pretreated with different concentrations of Cap(1,3,10 μM)and CAPZ(1,3,10 μM)were added to incubate for 0,4,8,and 12 h,and the formation of vascular network was observed under the microscope.(5)The effects of Cap and CAPZ on the ADSCs migration were explored: ADSCs were incubated in low serum medium with different concentrations of Cap(0,1,3,10μM)and CAPZ(10 μM)for 24 h.Wound healing was used to test the degree of scratch closure,and transwell chamber was used to measure the number of cells migrating across membrane.(6)A wound model was constructed in normal rats,and the wounds were treated with PBS,ADSCs,and Cap pretreated ADSCs,respectively.The wound tissue was photographed at 0,3,7,10 and 14 days after surgery to observe the healing.The wound tissue on the 7th and 14 th days was embedded in paraffin and sectionalized,and the reepithelialization and vascularization of the wound were observed by HE staining and immunohistochemistry.(7)The type II diabetic rat model was established by feeding male SD rats with high-fat and high-sugar diet combined with intraperitoneal injection of streptozotocin solution,and set up groups: normal group(Normal),diabetes group(DM),dietary Capsaicin-supplemented diabetes group(Cap+DM).The primary adipose stem cells(ADSCs,DM-ADSCs and Cap+DM-ADSCs)were extracted from the rats in each group by enzymatic digestion,and the endothelium was induced at the P3 passage for14 days.The expression of vascular endothelial marker protein CD31 in ADSCs was detected by Western blotting.(8)A wound model was constructed in diabetic rats,and ADSCs,DM-ADSCs,and Cap+DM-ADSCs were used to treat the wounds respectively.The wound tissue was photographed at 0,3,7,10 and 14 days after operation to observe the healing.Results:(1)Flow cytometry indicated that ADSCs of Sprague-Dawley rats were positive expression of CD29,CD90,and negative expression of CD31,CD45.(2)ADSCs were successfully differentiated into endothelial cells,and the cell morphology changed from shuttle to cobblestone-like circle.Western blotting results showed that the expression of CD31 was obviously increased.Cap significantly promoted the endothelial differentiation of ADSCs,and the expression of CD31 protein was significantly increased,while CAPZ inhibited the endothelial differentiation of ADSCs and down-regulated the expression of CD31 protein obviously.(3)Under normal sugar environment,Cap promoted the secretion of VEGF,b FGF and NO in ADSCs,while CAPZ inhibited the secretion of VEGF,b FGF and NO in ADSCs.Under high glucose environment,Cap improved the secretion of VEGF in ADSCs,while had little effect on b FGF.High glucose treatment significantly decreased the secretion of NO in ADSCs,and Cap restored it.(4)The results of Matrigel two-dimensional network forming experiment showed that ADSCs-CM promoted the formation of HUVECs vascular network.It increased the cross number and branch length significantly.Cap pretreament increased the number of cross points and branch length of HUVECs vascular network significantly,CAPZ pretreament inhibited the formation of HUVECs vascular network in a dosedependent manner.Cap(3μM)significantly increased the number of cross points and branch length of HUVECs vascular network,and maintained the formation of HUVECs vascular network at 4h,8h,12 h.(5)The results of both wound healing and transwell chamber experiments showed that Cap(1μM,3μM,10μM)significantly reduced the scratch area and increased the number of transmembrane migrating cells in a dose-dependent manner,and CAPZ(10μM)significantly inhibited the migration of ADSCs.(6)The results of wound healing in normal rats showed that Cap pretreatment enhanced the effect of ADSCs on wound healing in normal rats.HE staining and CD31 immunohistochemistry results showed that Cap pretreatment promoted the early epithelialization of wound and increased the thickness of new wound epithelium in normal rats.Cap-ADSCs group exhibited more CD31 positive cells in wound.(7)Western blotting results showed that compared with normal rat ADSCs,the expression of CD31 protein in DM-ADSCs was significantly decreased after endothelial induction,and the expression of CD31 protein in Cap+DM-ADSCs was recovered.The wound healing results of diabetic rats showed that DM-ADSCs had poor ability to promote diabetic wound healing,while dietary Cap improved the wound healing effect of DM-ADSCs in diabetic rats.Conclusion:(1)The TRPV1 agonist Cap can promote endothelial differentiation,paracrine pro-angiogenic factors and migration ability of ADSCs,thereby enhancing the ability of ADSCs to promote wound healing in normal rats.(2)Cap can improve the ability of ADSCs to differentiate into endothelial cells and secrete angiogenic factors in high glucose environment,thus enhancing the effect of ADSCs on wound healing in diabetic rats. |
PDF Full Text Request