The Role Of 3,3'-Diindolylmethane Pretreated Human Umbilical Mesenchymal Stem Cells In Cutaneous Wound Healing And The Mechanism

Objective: Skin serves as the largest organ of human to defend infection and invation outside. Skin burn injury destroys the protective barrier and brings patient dysafia and pain. However, the curative effect of existing therapies including mesenchymal stem cell-based cell therapy is unsatisfactory. The crossover of small modecular drug and stem cell fate regulation benefits the wound healing and regenerative medicine. In this research, 3,3'-diindolylmethane(DIM) was selected to pre-treat hucMSCs for a better therapy in wound healing and discover the potential molecular mechanism.Methods: The concentration and working time duration of DIM was decided by MTT assay. A deep second-dagree burn injury rat model was established to verify the therapeutic effect of hucMSCs and DIM pre-treated hucMSCs(DIM-hucMSCs).HE staining and wound score was used to judge the epithelial regeneration and the recovery of dermal appendages. Immunohistochemical staining of PCNA was detected to reflect the proliferation of skin cells.CK19 expression and Collegen I/III ratio was respectively tested by immunofluorescence and real-time PCR.The expression level of sternness transcription factor Oct4,Sox2,Nanog,Sall4 was detected by Western blot and real-time PCR. Colony forming assay was used to verify the self-proliferation of DIM-hucMSCs. The multi-differetiation capacity was detected by adipogenic indction and osteogenic indution. Luminex assay reflected the paracrine capacity of DIM-hucSMCs. When looking for the potential mechanism of DIM treatment on hucMSCs,luciferase reporter activity was tested and the expression level of ?-catenin was detected by Western blot.Western blot was also used to reflect the expression of ?-catenin target protein CD44,CyclinD3. The (3-catenin signalling inhibitor ICG001 was used to block the (3-catenin activation. The colony- foming assay, adipogenic indction and osteogenic indution were all carried out to confirm the fuction of Wnt/?-catenin activation on DIM-hucMSCs.The therapeutic effect of DIM-hucMSC after ?-catenin inhibition was also verified in deep second-dagree burn injury rat model.To further find out the specific Wnt/Wnts, real-time PCR was used to screen all Wnt family members. Lentivirus interference effeciency of Wntll was detected by real-time PCR and Western blot. The colony foming assay, adipogenic indction and osteogenic indution were used to confirm the fuction of Wntll on Wnt/?-catenin actvation. The in vivo model was also used to investigate the therapeutic effect of Wnt11 knock down DIM-hucMSCs.The conditioned medium of hucMSCs and DIM-hucMSCs were collected,and ELISA assay was used to detect the Wntll secretion level in the conditioned medium.HucMSC and DIM-hucMSC derived exosmes were isolated by sucrose density gradient centrifugation. The surface markers of exosomes, as CD63, CD81 and CD9 were determined by Western blot and the diameter,distribution and particle numbers were analyzed by Nanosight.The colony-forming assay and scratch assay were used to detect the fuction of DIM-hucMSC derived exosomes and the exosome-free conditioned medium.Results: The MTT assay showed hucMSC were more tolerant than gastric cancer cells when pre-treated with DIM,the IC50 was 158.11?M. We selected 50?M as our experimental concentration which exerted the strongest effects as well as not impair the cell viability. DIM-hucMSCs showed a better recovering role than hucMSCs in rat deep-second degree burn injury. The scabs had already sloughed away and the new skin tissues exposed in DIM-hucMSC treated rats.The hair follicle and other skin appendages were repaired.The expression of PCNA,CK19 and Collegen ?/? were also higher in DIM-hucMSC treated group. In vitro,DIM could upregulate the expressrion of stemness transcription factors as Oct4, Sox2,Nanog,Sall4 in both mRNA and protein level. The colony-forming assay and adipogenic indction,osteogenic induction indicated DIM-hucMSC possessed a better self-proliferation capacity and were more likely to be induced into lipoblast and osteoblast.Luminex showed a increased paracrine of cytokines and chemokines of DIM-hucMSCs.The luciferase reporter activity assay showed a higher Wnt activity in DIM-hucMSCs. The?-catenin inhibitor ICG001 reversed the Wnt activity and the expression of p-catenin in DIM-hucMSCs. It also inhibited the DIM caused sternness upregulation and the higher paracrine capacity of hucMSCs. ICG001 treated DIM-hucMSCs lost their advantages in wound healing. The Wntll knockdown inhibited ?-catenin activation and stemness induction in DIM-hucMSCs and abrogated their therapeutic effects in vivo.ELISA and Western blot indicated that exosomes are effective transporters for Wnt11.Conclusion: Our findings indicate that DIM upregulates the stemness and paracrine capacity of hucMSCs via increased exosomal Wnt11 secretion,which provides a novel strategy for improving the therapeutic effects of hucMSCs on wound healing.