Effect And Mechanism Of Stem Cells From Human Exfoliated Deciduous Teeth Transplantation Combined With Hyperbaric Oxygen In Treating Type 2 Diabetic Rats

Diabetes mellitus（DM）is a group of metabolic diseases characterized by hyperglycemia and/or microvascular and macrovascular diseases.Type 2 diabetes mellitus（T2DM）is the main type of DM which is caused by a variety of causes including insulin resiststance and insulin secretion defects.Its etiology is complex and is difficult to control and cure.Stem cells are the initial sources of various tissue cells in the body,and their most remarkable biological feature is self-renewal and multidirectional differentiation.Human deciduous tooth pulp mesenchymal stem cells have a wide range of sources and they have the ability of proliferation and differentiation of primitive cells and the function of paracrine cytokines.Their immunogenicity is low,and there is no ethical controversy.They can be homing by their chemotaxis in vivo.They may reduce islet apoptosis,promote islet proliferation and transdifferentiation,enhance islet function and improve insulin resistance through various ways.They may become seed cells for the treatment of T2DM.Hyperbaric oxygen therapy improves oxygen supply of local tissue,boosts metabolism,and may modulate immunity.It may be synergistic with stem cell therapy.In this study,dental pulp stem cells transplantation combined with hyperbaric oxygen therapy was performed in type 2 diabetic rats,in order to provide a theoretical basis for the clinical application of stem?cells?from?human?exfoliated?deciduous?teeth in the treatment of type 2diabetes.[Objective]To investigate the therapeutic effect of stem?cells?from?human?exfoliated?deciduous?teeth transplantation combined with hyperbaric oxygen on type 2 diabetic rats,and to explore the possible mechanism[Methods]After adaptive feeding 4-week-old SD rats for 1 week,high-fat diet was given for 8weeks,and then intraperitoneal injection of 30mg/kg streptozotocin（STZ）was given to induce the model of type 2 diabetic rats.After successful modeling,diabetic rats were randomly divided into stem cell therapy group,hyperbaric oxygen therapy group,stem cell combined hyperbaric oxygen therapy group and diabetes control group,and a normal rat control group was set up at the same time.The intake,weight and blood glucose of rats were monitored weekly.OGTT assay was performed in rats before and after 8 weeks high-fat feeding on normal rats,before and after 2,4,and 6 weeks treatment for diabetic rats.Blood glucose and islet function were evaluated.The area under the curve of blood glucose and insulin(AUC_0-120min)and homeostasis model assessment of insulin resistance（HOMA-IR）were calculated.Serum lipid,24-hour urine protein,tumor necrosis factorα（TNF-α）and interleukin-6（IL-6）were measured.After 6 weeks treatment rats were sacrificed,HE staining of pancreas,lung,liver and kidney was observed under light microscope.The morphological changes of islet cells were observed by electron microscopy.Immunohistochemistry and immunofluorescence staining of insulin and glucagon in islets of rats were observed.Image-pro Plus 6.0software was used to calculate the staining intensity of insulin and glucagon in immunohistochemical staining of islet tissue.Islet cells TUNEL apoptosis staining and Ki67/PCNA proliferation staining were performed.Apoptosis index and proliferation index were calculated.Quantitative real-time polymerase chain reaction assay（qRT-PCR）was used to detect the expression of Pdx1,Ngn3 and Pax4 mRNA in pancreatic tissue.SPSS 22.0 software was used for statistical analysis of the data,and the treatment results were compared among the groups.[Results]1.Compared with the normal control group,after 8 weeks of high-fat feeding,blood glucose,HOMA-IR,serum triglycerides（TG）and cholesterol（TC）levels,serum TNF-αand IL-6 levels in the high-fat diet group were all increased,with significant weight gain（all p<0.05）.There was no significant difference with 24-hour urine protein（p>0.05）;2.Compared with the diabetes control group,the AUC_0-120min-120min of blood glucose,serum TNF-αand IL-6 levels in the stem cells combined with hyperbaric oxygen therapy group were decreased after 2 weeks treatment.Body weight gained and fasting blood glucose decreased after 3 weeks.After 4 weeks,HOMA-IR,serum TG and 24-hour urine protein were all decreased,and AUC_0-120min of insulin was increased.Serum LDL cholesterol（LDL-C）decreased after 6 weeks（all p<0.05）;3.Compared with the diabetes control group,after 2 weeks treatment,serum TNF-αwas decreased in the stem cell therapy group.After 4 weeks treatment,fasting blood glucose,AUC_0-120min-120min of blood glucose,HOMA-IR,serum TG,24-hour urine protein level,serum IL-6 were decreased,and AUC_0-120min of insulin were increased.After 6 weeks body weight gained and serum LDL-C decreased（all p<0.05）;4.Compared with the diabetes control group,after 4 weeks treatment serum TG and TNF-αdecreased in the hyperbaric oxygen therapy group,and after 6 weeks serum LDL-C and 24-hour urine protein decreased（all p<0.05）.5.Compared with the diabetes control group after 6 weeks treatment,the number of islets in the pancreas increased,morphology of islets was improved,and inflammation of islets was relieved both in the stem cell combined with hyperbaric oxygen therapy group and the stem cell therapy group.Immunohistochemistry and immunofluorescence showed that the number of insulin-positive cells（beta cells）increased and the number of glucagon positive cells（alpha cells）decreased in the islets of the two groups（p<0.05）.A small number of insulin-positive cells were found in the glomeruli and renal stroma of the combined therapy group and stem cell therapy group.6.Compared with the diabetes control group after 6 weeks treatment,TUNEL apoptosis index of islet cells was significantly decreased in the combined therapy group and the stem cell therapy group,and the proliferation index of Ki67 and PCNA were significantly increased（all p<0.05）.The index level of combined therapy group was better than that of stem cell therapy group or hyperbaric oxygen therapy group（all p<0.05）.7.Compared with the diabetes control group after 6 weeks treatment,the mRNA expression levels of Pdx1,Ngn3 and Pax4 in pancreatic tissues were significantly increased in the combined therapy group and the stem cell therapy group（all p<0.05）.The expression level in the combined therapy group was higher than that in the stem cell therapy group or the hyperbaric oxygen therapy group（all p<0.05）.[Conclusion]Stem cells from human exfoliated deciduous teeth transplantation combined with hyperbaric oxygen therapy could treat T2DM in rats.It could reduce blood glucose,increase insulin level,improve insulin resistance,reduce blood lipid（TG and LDL-C）,and reduce 24-hour urine protein level.Increase of insulin may be achieved by increase of the mRNA expression of Pdx1,Pax4 and Ngn3 genes in the pancreas,thereby inducing pancreatic islets differentiation and transdifferentiation,reducing inflammatory reactions and apoptosis.It is possible that some of these stem cells may locate in the pancreas and kidneys,and may directly differentiate into insulin-producing cells.Improvement of insulin resistance may be achieved by regulating immunity to reduce TNF-αand IL-6,the pro-inflammatory cytokines,thereby reducing the body’s inflammatory response.Changes in blood lipid and urine protein may be associated with improved inflammatory status.The effect of stem cells combined with hyperbaric oxygen therapy was faster than that of stem cell transplantation alone and better than that of hyperbaric oxygen therapy alone.Combination of hyperbaric oxygen therapy has a synergistic effect on the efficacy of stem cell transplantation.