| Diabetes(Diabetes Mellitus,DM)is a global chronic metabolic disease.According to the International Diabetes Federation(IDF)report,the population of diabetes patients will increase from 415 million in 2015 to 642 million in 2040.Type 1 Diabetes at an annual rate of 3%to 5%of cases increased steadily.The number of young people(<20 years)with DM worldwide has exceeded 1 million.According to the current trend,100,000 more people are expected each year.Insulin lifetime replacement therapy is the main clinical treatment method for T1DM at present,but it cannot prevent the progressive failure of islet function,nor can it effectively prevent the occurrence and progression of T1DM related complications.In recent years,stem cell therapy is expected to protect or even rebuild the endogenous insulin secretion system through the replacement or regeneration of islet?cells,thus improving the disease course and prognosis,which has become a focus of research in the field of T1DM treatment.The application of stem cells from pulps of human exfoliated deciduous teeth(SHED)gradually shows its potential advantages.Compared with other mesenchymal stem cells,SHED has abundant sources,simple acquisition,strong proliferation ability,low immunogenicity,low tumorigenic risk,and no ethical problems.It has better immunomodulatory effect,better biological stability and benign characteristics,and lower apoptosis and senility.Shed has been shown to be induced into islet beta cells,showing great potential for future use in the treatment of T1DM.At present,the research on its application in DM is gradually developing,and there have been no reports on the basic and clinical studies related to its recovery of?-cell function in treating T1DM,and the mechanism of its action remains to be clarified.[Objective]1.SHED was transplanted through caudal vein and dorsal pancreatic artery to explore the effect of different grafting approaches on SHED in STZ-induced DM rats.2.Subcutaneously injected insulin to maintain blood glucose in DM rats at different levels.Repeated transplantation of SHED through tail vein was conducted to investigate the difference in efficacy of repeated infusion of SHED in DM rats with different blood glucose levels.3.Through animal experiments and cell experiments,the possible mechanism of SHED in the treatment of DM was explored.[Methods]Male SD rats(180-200g)were given 60mg/kg STZ after adaptive feeding to establish a diabetes model.1.SHED was used to treat DM rats by different infusion routesForty-eight STZ-induced DM rats were randomly selected and divided into diabetes mellitus control group(DM group,n=14),Caudal vein infusion SHED group(CV group,n=16)and Dorsal pancreatic artery infusion SHED group(DPA group,n=18),12 control rats were normal control group(NC group,n=12).After the formation of the model,the CV group and the DPA group were given subcutaneous insulin therapy for decrease blood glucose,and the stem cell therapy was started after reaching the target blood glucose level about one week,and the insulin therapy continued until the end of the treatment.The food intake,body weight and fasting blood glucose of rats were monitored weekly,the random blood glucose of rats in CV group and DPA group was monitored daily,and the insulin injection dose was recorded.Before treatment and 2 weeks after SHED treatment,Oral glucose tolerance test(OGTT)was performed to evaluate glucose metabolism,and serum samples were collected to detect C-peptide and glycated serum protein(GSP).After 2weeks of stem cell treatment,the rats were sacrificed and the organs were collected for follow-up study.2.SHED was used to treat DM rats with different blood glucose levelsThirty-six STZ-induced DM rats were divided into diabetes mellitus control group(DM group,n=6),insulin treatment group(n=12)and stem cell treatment group(n=18),the insulin treatment group was divided into Middle glycemic group(MG group)and Low glycemic group(LG group).Meanwhile,The stem cell treatment group was divided into High glycemic SHED group(HGSHED group),Middle glycemic SHED group(MGSHED group)and Low glycemic SHED group(LGSHED group),with 6 rats in each group.The rats without model were normal control group(NC group,n=6).Rats in the MG,MGSHED,LG and LGSHED groups were all given insulin therapy.Stem cell therapy was started after the target blood glucose was reached within 2 weeks,and insulin therapy continued until the end of treatment.Food intake,body weight and fasting blood glucose were monitored every week,random blood glucose of rats in the MG,MGSHED,LG and LGSHED groups was monitored every day,and insulin injection volume was recorded.Before treatment and 2 weeks after the third infusion of stem cells,OGTT test was performed on all rats to evaluate glucose metabolism,and serum samples were collected to detect C-peptide,GSP and so on.After 5 weeks of stem cell treatment,the rats were sacrificed and the organs were collected for follow-up study.3.Study on the mechanism of SHED in treating DM ratsThe pathological changes of pancreas were observed by hematoxylin and eosin staining.The internal structure changes of?cells were observed by transmission electron microscopy.The?/?islet cell ratio and the secretion of insulin and glucagon in islet cells were investigated by immunohistochemistry and immunofluorescence double labeling.Reactive Oxygen Species(ROS)staining in frozen sections and oxidative stress related indicators were detected to evaluate the oxidative stress in the pancreas of rats in each group.The KEAP1/Nrf2 antioxidant signaling pathway,the activation of p-ERK and the expression of anti-apoptotic protein Bcl-2 were analyzed by Western-blot(WB).4.Effect of SHED on improving STZ-induced pancreatic islet Rin-M5F cell injury and its mechanism(1)Construction of cell modelDifferent concentrations of STZ(50mM/20mM/10mM/7.5 mM/5mM/2.5mM/1.25mM/1mM)were used to treat pancreatic islet Rin-M5F cells for different times(2h/6h/12h/24h).Cell-Counting Kit-8 detection was performed and the inhibition rate of50%was selected as the concentration and time for the subsequent cell experiment to build the model.ROS was detected at this concentration and time to confirm the successful construction of DM oxidative stress cell model.(2)Stimulation experiment of SHED culture supernatantAccording to different treatments,they were divided into normal control group(NC group),SHED culture medium treatment group(CM group),STZ group and STZ+SHED culture medium treatment group(STZ+CM group).ROS fluorescence staining and flow cytometry,oxidative stress related indexes,apoptosis flow cytometry,EDU-594 Cell proliferation fluorescence staining and flow cytometry were performed for each group.The m RNA expression levels of KEAP1/Nrf2 pathway,anti-apoptotic protein Bcl-2,apoptosis-related proteins Bax and Caspase-3,Proliferating Cell Nuclear Antigen(PCNA)were determined by fluorescence quantitative PCR.(3)Key role cytokine determination and siRNA interferenceLiterature review was confirmed that HGF was a cytokine in this study.The improvement of STZ-induced?-cell function in DM islets by SHED may be closely related to the higher secretion of HGF.The HGF gene of SHED was interfered by siRNA.(4)The comparative experiment conducted between SHED culture medium and siRNA-HGF SHED culture mediumAccording to different treatments,they were divided into STZ group,STZ+SHED culture medium treatment group(STZ+CM group),STZ+siRNA interference SHED culture medium treatment group(STZ+siRNACM group)and STZ+recombinant hepatocyte growth factor treatment group(STZ+HGF group).Perform relevant tests for each group,including ROS fluorescence staining and flow cytometry analysis of related parameters of oxidative stress tests,flow cytometry to detect apoptosis,Ed U-594 Cell proliferation fluorescence staining and flow cytometry detection.The m RNA expression levels of Nrf2/Keap1 pathway,anti-apoptotic protein Bcl-2,apoptosis-related proteins Bax and Caspase-3,and PCNA were detected by fluorescence quantitative PCR.Furthermore,the effects of HGF on Keap1/Nrf2 signaling pathway,p-Erk,p-Akt and apoptosis-related proteins in the cell model were further investigated by Western Blot.[Results]1.SHED was used to treat DM rats by different infusion routesAfter stem cell treatment,the average weekly daily food intake in CV group and DPA group were significantly lower than that in DM group(both P<0.01),and CV group was lower than that in DPA group(P<0.01).The body weight of rats in CV group and DPA group were significantly higher than that in DM group(P<0.01)(P<0.05),and the weight of rats in CV group was also significantly higher than that in DPA group(P<0.01).One week after treatment,the mean value of fasting blood glucose in both groups were the same(15.68mmol/L),and 2 weeks after treatment,the fasting blood glucose in both groups were significantly lower than that in the DM group(all P<0.01).The average daily insulin injection volume of CV group and DPA group were significantly lower than those before treatment(Week 5)(P=0.001,P=0.001).After treatment,AUC0-120min of blood glucose in CV group and DPA group were significantly lower than that in DM group(both P<0.01).The AUC0-120min levels of C-peptide in CV group and DPA group were significantly higher than those before treatment(P=0.004)(P=0.001),and CV group was significantly higher than those in DM group and DPA group(both P<0.01).The level of GSP in DM group was significantly higher than those in NC group,CV group and DPA group(all P<0.01),but there were no significant difference among the three groups(P>0.05).There was no significant change in the GSP of the CV group before and after treatment(P=0.309),while the GSP of the DPA group was lower than that before treatment(P=0.001).2.SHED was used to treat DM rats with different blood glucose levelsAfter stem cell treatment,the average daily food intake of rats in MGSHED,LG and LGSHED groups were significantly lower than those in DM,HGSHED and MG groups(P<0.05)(P<0.01).The body weight of rats in MG group,MGSHED group,LG group and LGSHED group were significantly higher than those in DM group and HGSHED group(all P<0.01).The fasting blood glucose of rats in the MG group,MGSHED group,LG and LGSHED group were significantly lower than those in the DM group and HGSHED group in the same period,and there were no significant difference in the fasting blood glucose among the MG,MGSHED,LGSHED and NC groups(P>0.05).The average daily insulin dose in MG,MGSHED,LG and LGSHED groups were significantly decreased compared with those before stem cell treatment(P<0.05)(P<0.01).After treatment,AUC0-120min of blood glucose in MG,MGSHED,LG and LGSHED groups decreased gradually compared with those before treatment(all P<0.01),and was significantly lower than that in DM group and HGSHED group(all P<0.01).AUC0-120min of C-peptide in DM group was significantly lower than that before treatment(P<0.01),while there was no significant change in HGSHED group before and after treatment.The AUC0-120min level of C-peptide in DM group,HGSHED group and MG group were significantly lower than that in NC group(P<0.05)(P<0.01);AUC0-120min level of C-peptide in MG group,MGSHED group,LG group and LGSHED group were significantly higher than those in DM group and HGSHED group(P<0.01).After treatment,GSP levels in MG,MGSHED,LG and LGSHED groups were significantly lower than those in DM group and HGSHED group(all P<0.01).3.Study on the mechanism of SHED in treating DM rats(1)Compared with DM group and HGSHED group,the volume of islet in MG group,MGSHED group,LG group and LGSHED group were significantly increased,the number of islet cells were significantly increased,the definition of nuclear staining was significantly increased,vacuolar cells and hyalinosis were decreased,and the infiltration of inflammatory cells were significantly reduced.The volume of MGSHED group was larger than that of MG group,and LGSHED group than that of LG group,the improvement were more obvious.Among them,the volume of islets in the MGSHED group was the largest,but smaller than that in the NC group.(2)The results of pancreatic electron microscopy showed that the state of islet?cells were improved in different degrees in each treatment group,and the improvement was most obvious in MGSHED group.Islet immunohistochemistry and fluorescence both showed that the ratio of?/?cells increased,insulin secretion increased,and glucagon decreased in different degrees in each treatment group.(3)For ROS staining,the average fluorescence intensity in the DM group was the highest,and the average fluorescence intensity in each treatment group were reduced to a certain extent compared with that in the DM group,among which the average fluorescence intensity in the MGSHED group was the lowest.(4)Pancreas total antioxidant capacity(T-AOC),superoxide dismutase(SOD)and glutathione(GSH)increased to different degrees,while malondialdehyde(MDA)and nitric oxide synthase(NOS)type,especially i NOS,decreased to a certain extent in treatment groups.(5)Western Blot analysis showed that the expression of Nrf2 protein in the pancreas of rats in the DM group was significantly lower than that in the NC group,and the expression of Nrf2 protein in all treatment groups were increased to a certain extent,among which the MGSHED group increased the most.The expression of Keap1 protein in pancreas of rats in DM group was significantly higher than that in NC group,and the expression of Keap1 protein in all treatment groups were decreased to a certain extent,among which the MGSHED group was the least.The p-Erk,the anti-apoptotic protein Bcl-2 were significantly decreased in DM group,and increased to a certain extent in all treatment groups.The MGSHED group was significantly higher than the MG group,and the LGSHED group was significantly higher than the LG group.4.Effect of SHED on improving STZ-induced pancreatic islet Rin-M5F cell injury and its mechanism(1)The concentration of STZ was 5mM and the time was 12h,which was used as the modeling conditions for subsequent cell experiments.The fluorescence intensity of ROS detection was significantly enhanced,indicating that the oxidative stress cell model was successfully constructed.(2)Stimulation experiment of SHED culture mediumIn STZ+CM group,compared with STZ group,the average ROS fluorescence intensity decreased,antioxidant related indexes T-AOC,SOD and GSH increased,lipid peroxidation product MDA decreased,cell apoptosis rate decreased and cell proliferation rate increased significantly(P<0.01).The m RNA expression of Nrf2 in STZ+CM group was significantly higher than that in STZ group(P<0.01),while the expression of Keap1 in STZ+CM group was significantly lower than that in STZ group(P<0.01).The m RNA expression levels of Bcl-2 and PCNA in STZ+CM group were significantly higher than those in STZ group(P<0.01),while Bax and Caspase-3 were significantly lower than those in STZ group(P<0.01).(3)Key role cytokine determination and siRNA interferenceWe used siRNA-HGF to interfer with SHED,and HOMO-HGF-2 significantly inhibited HGF m RNA(inhibition rate was about 71.78%)(P<0.01)and HGF protein expression in SHED cells.The concentration of HGF in SHED culture medium detected by ELISA was significantly lower in siRNA-HGF2 group than that in NC group and siRNA-NC group(P<0.05)(P<0.01).(4)The comparative experiment was conducted between SHED culture medium and siRNA-HGF SHED culture mediumThe average ROS fluorescence intensity in STZ+CM group was significantly lower than that in STZ+siRNACM group(P<0.01).T-AOC in STZ+siRNACM group was significantly lower than that in STZ+CM group(p<0.01),cell proliferation rate(p<0.05),NRF2(p<0.01),Bcl-2,PCNA m RNA expression were significantly lower than those of STZ+CM group(p<0.01),but the m RNA expression of caspase-3 was significantly higher than that in STZ+CM group(p<0.01).(5)Western Blot analysis confirmed the proteins associated with the action of HGF,and it was found that the recombinant HGF could activate Nrf2,p-Erk and p-Akt,down-regulate Keap1,increase the expression of anti-apoptotic protein Bcl-2,and decrease the expression of apoptotic protein Caspase-3.[Conclusions]1.Different infusion routes and different blood glucose levels,SHED could both improve the symptoms,glucose metabolism related indexes and islet secretion function of DM rats to different degrees.2.Different transfusions transplanted SHED with the same number of transfusions,at the same glucose metabolism level and observed for the same time showed similar improvement in blood glucose.3.The therapeutic effect of SHED grafted by tail vein for many times on DM rats with different blood glucose levels was different.The overall improvement of symptoms,glucose metabolism and islet function of DM rats in the MGSHED group was better,indicating that maintaining a stable control of blood glucose at an appropriate level is more conducive to the survival and migration of SHED in vivo and the secretion of related cytokines,so as to achieve a better therapeutic effect.4.After controlling blood glucose level,multiple intravenous transplantation of SHED could significantly improve diabetic symptoms,reduce fasting blood glucose level,improve islet storage and secretion function,reduce insulin dose,and the effect of three transplants was better than that of once.5.SHED transplantation in diabetic rats might activate the endogenous oxidative stress system of the pancreas,enhance its antioxidant capacity,and oxidative stress regulation may play an important role.The most important antioxidant signaling pathway Keap1/Nrf2 was closely related to regulation.Moreover,the cell proliferation associated with p-Erk activation and the activation of Bcl-2 family proteins were related to the reduction of cell apoptosis.6.The vitro studies showed that SHED could reduce oxidative stress induced by STZ and promote the survival of pancreatic Rin-M5F cells,which was closely related to the secretion of a large amount of HGF.It might activate p-Erk,p-Akt and Keap1/Nrf2antioxidant signaling pathways through HGF,and activate endogenous antioxidant pathways,thereby affecting oxidative stress related indexes,reducing apoptotic proteins,and promoting the expression of anti-apoptotic and proliferation-related proteins to protect?-cells,which play an important role in anti-diabetic effects. |
PDF Full Text Request