Protective Effects on Melphalan-Induced Mouse Intestinal Injuries Treated By Human Umbilical Cord-Derived Mesenchymal Stem Cells

Introduction Autologous peripheral blood stem cell transplantation is front-line therapy with high-dose melphalan (HDM) for multiple myeloma (MM). Patients with MM who receive HDM usually suffer from severe gastrointestinal symptoms. It is an urgent demand to develop an effective method to repair the intestinal injuries. Therefore, the protective effects of human umbilical cord derived-mesenchymal stem cells (MSCs) are investigated in mouse model with melphalan-induced intestinal injuries.

Method Intestinal injury mouse model is developed in C57BL/6 intraperitoneally injected with 20mg/kg melphalan. The mice were divided into six groups: Group I, (treated with saline solvent), Groups II (with melphalan), Group III (with MSCs 24 hours prior to melphalan), Groups IV (infusion of MSCs 24 hours after melphalan). Groups V (exosomes from MSCs injected 24 hours before melphalan), Groups VI (MSCs exosomes injected 24 hours after melphalan). Each mouse received a total 1×10⁶ MSCs or 1×10⁹ particles of exosomes by injection through tail vein. The protective effects of MSCs on melphalan-induced intestinal injuries were assessed by mouse body weight, small bowel length, intestinal tissue damages, and overall survival (OS). When mice with a body weight drop of 18%-20% are given euthanasia. The followed indicators responsible for intestinal injuries and repairing of intestinal tissues were investigated including the pathological features with hematoxylin-eosin staining (HE), apoptosis with TUNEL, the proliferation by Ki67. The transcription expression of pro-inflammatory factors were analyzed by qPCR with intestinal tissues.

Results In this study, after injection of melphalan body weight of mice began to drop in Group II compared with Group I at 4 days. With infusion of MSCs, the body weights were increased in Group III and in Group IV. Furthermore, 80% of mice developed diarrhea after 4 days of melphalan, but no mouse developed diarrhea when infusion of MSCs. The death occurs in 100% of mice after 6-8 days in Group II, whereas, all mouse survived in Group I. After treatment with MSCs, 88% mice survived in Groups III, and 100% mice survived in Group IV. To compare the length of small intestine, 6 mice were sacrificed in each group on day 4. Small intestine length in Group II was shorter than Group I, while the length with MSCs treatment significantly increased in Group III and in Group IV. The structural integrity of small intestinal epithelium was evaluated by HE staining. The length of small intestinal villus and the depth of the small intestine crypts in Group II was shortened in contrast to Group I. After MSCs, the structural integrity significantly improved by increases in both crypt depth and villus height in Group III and in Group IV. With application of immunohistochemistry, TUNEL-positive cells in intestinal epithelial cells increase in Group II, and Ki67-positive cells decrease compared with Group I. With infusion of MSCs, TUNEL-positive cells decrease in Group III and in Group IV, Ki67-positive cells increase in Group III and in Group IV. After treatment with melphalan, pro-inflammatory cytokines IL-6 and TNFα are increased by comparison with Group I. However, MSCs downregulated the expression of IL6 and TNFα in Group III and in Group IV. To further explore how MSCs play a restorative function in repairing intestinal tissues injuries, infusion of MSCs exosomes were certified to have restorative function, mainly to improve OS, to promote the proliferation of small intestinal epithelial cells, and to inhibit the elevation of inflammatory cytokines.

Discussion MSCs repaired the melphalan-induced injuries in intestine tissues, and prolonged OS of mice treated with melphalan. The exosomes secreted from MSCs partly played a key role in repairing melphalan-induced intestinal tissue injuries.

Keywords Melphalan, Human umbilical cord, mesenchymal stem cells, intestinal injury Introduction Autologous peripheral blood stem cell transplantation is front-line therapy with high-dose melphalan (HDM) for multiple myeloma (MM). Patients with MM who receive HDM usually suffer from severe gastrointestinal symptoms. It is an urgent demand to develop an effective method to repair the intestinal injuries. Therefore, the protective effects of human umbilical cord derived-mesenchymal stem cells (MSCs) are investigated in mouse model with melphalan-induced intestinal injuries.

Method Intestinal injury mouse model is developed in C57BL/6 intraperitoneally injected with 20mg/kg melphalan. The mice were divided into six groups: Group I, (treated with saline solvent), Groups II (with melphalan), Group III (with MSCs 24 hours prior to melphalan), Groups IV (infusion of MSCs 24 hours after melphalan). Groups V (exosomes from MSCs injected 24 hours before melphalan), Groups VI (MSCs exosomes injected 24 hours after melphalan). Each mouse received a total 1×10⁶ MSCs or 1×10⁹ particles of exosomes by injection through tail vein. The protective effects of MSCs on melphalan-induced intestinal injuries were assessed by mouse body weight, small bowel length, intestinal tissue damages, and overall survival (OS). When mice with a body weight drop of 18%-20% are given euthanasia. The followed indicators responsible for intestinal injuries and repairing of intestinal tissues were investigated including the pathological features with hematoxylin-eosin staining (HE), apoptosis with TUNEL, the proliferation by Ki67. The transcription expression of pro-inflammatory factors were analyzed by qPCR with intestinal tissues.

Results In this study, after injection of melphalan body weight of mice began to drop in Group II compared with Group I at 4 days. With infusion of MSCs, the body weights were increased in Group III and in Group IV. Furthermore, 80% of mice developed diarrhea after 4 days of melphalan, but no mouse developed diarrhea when infusion of MSCs. The death occurs in 100% of mice after 6-8 days in Group II, whereas, all mouse survived in Group I. After treatment with MSCs, 88% mice survived in Groups III, and 100% mice survived in Group IV. To compare the length of small intestine, 6 mice were sacrificed in each group on day 4. Small intestine length in Group II was shorter than Group I, while the length with MSCs treatment significantly increased in Group III and in Group IV. The structural integrity of small intestinal epithelium was evaluated by HE staining. The length of small intestinal villus and the depth of the small intestine crypts in Group II was shortened in contrast to Group I. After MSCs, the structural integrity significantly improved by increases in both crypt depth and villus height in Group III and in Group IV. With application of immunohistochemistry, TUNEL-positive cells in intestinal epithelial cells increase in Group II, and Ki67-positive cells decrease compared with Group I. With infusion of MSCs, TUNEL-positive cells decrease in Group III and in Group IV, Ki67-positive cells increase in Group III and in Group IV. After treatment with melphalan, pro-inflammatory cytokines IL-6 and TNFα are increased by comparison with Group I. However, MSCs downregulated the expression of IL6 and TNFα in Group III and in Group IV. To further explore how MSCs play a restorative function in repairing intestinal tissues injuries, infusion of MSCs exosomes were certified to have restorative function, mainly to improve OS, to promote the proliferation of small intestinal epithelial cells, and to inhibit the elevation of inflammatory cytokines.

Discussion MSCs repaired the melphalan-induced injuries in intestine tissues, and prolonged OS of mice treated with melphalan. The exosomes secreted from MSCs partly played a key role in repairing melphalan-induced intestinal tissue injuries.

Keywords Melphalan, Human umbilical cord, mesenchymal stem cells, intestinal injury

No relevant conflicts of interest to declare.