Protective Effect And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cell-derived Exosomes On Kidney Of Diabetic Mice

Background:Diabetic nephropathy(DN)is one of the most serious microvascular complications of Diabetes mellitus(DM).Patients with DN are prone to develop end-stage renal disease(ESRD).These patients have poor quality of life,heavy economic burden,and need lifelong blood purification replacement therapy.Therefore,it is very important to explore the pathogenesis of DN and possible effective treatment methods.DM includes type 1 diabetes(T1DM),type 2 diabetes(T2DM)and special type diabetes,of which T1 DM and T2 DM are the most common types.The occurrence and development of both are related to inflammatory reaction in the body,and can induce DN.Nearly 30% of T1 DM patients will develop into DN,while the incidence rate of T2 DM patients is about 40%.In the pathogenesis of DN,systemic and renal tissue inflammation plays a very important role,and the inflammatory response in the kidneys determines the degree of renal injury progression.As the main innate immune cells in the body,macrophages play an important role in the progression of DN,and the degree of renal macrophage infiltration is significantly correlated with the progression of DN.Macrophages are mainly divided into two phenotypes: classically activated type 1 macrophages(M1)and alternatively activated type 2 macrophages(M2).In the inflammatory response of DN,macrophages have strong functional plasticity and regulate the inflammatory response by polarizing into different phenotypes.M1 macrophages generally promote the progression of inflammation in DN by upregulating the intensity of the inflammatory response,while M2 macrophages can alleviate their autoimmune response by downregulating the intensity of the inflammatory response.Therefore,searching for methods that can regulate the inflammatory response during DN by regulating changes in macrophage phenotype may be a very promising therapeutic strategy.XMesenchymal stem cells(MSC)have the potential to treat inflammatory diseases mainly due to their cell replacement and immune regulation functions.Exogenous MSC can accumulate locally in damaged tissues and repair tissues by cell replacement,while the immune regulation of MSC is mainly attributed to its paracrine mechanism.Mesenchymal stem cell‐derived exosomes(MSC-Exo)are easier to preserve than MSC,have stable biochemical activity,no potential toxicity,and have better safety in treating diseases.Mesenchymal stem cell exosomes have the function of influencing immune cell differentiation,which can inhibit inflammatory reaction at injury site and alleviate tissue injury by reducing proliferation of proinflammatory cells(such as monocytes,M1 macrophages,Th1 and Th17 cells)and increasing proportion of anti-inflammatory cells(such as M2 macrophages and Treg cells).At the same time,pretreatment of MSCExo with some inflammatory factors(TNF-α and IFN-γ)could enhance its immunomodulatory effect.Therefore,mesenchymal stem cell exosomes may be a very promising method for treating inflammatory damage in DN.It is currently unclear how MSC-Exo regulates the inflammatory response during the DN process,whether it can exert therapeutic effects on DN by regulating changes in macrophage phenotype,and whether pretreated MSC Exo with inflammatory factors can enhance this therapeutic effect.Therefore,elucidating the intrinsic mechanisms of action between MSC-Exo,macrophages,and DN,and providing basic research basis for new DN treatment strategies,is of great significance.Objective:To investigate whether MSC-Exo can regulate the changes of macrophage phenotype and alleviate the renal inflammation of DN,whether MSC-Exo pretreated with inflammatory factors can enhance the above effects,and to explore the possible molecular mechanisms,to provide theoretical support and experimental basis for transformation of DN into clinical treatment.Methods:Part 1 Isolation and identification of exosomes from umbilical cord mesenchymal stem cells1.Human umbilical cord-derived mesenchymal stem cells(HUC-MSC)were extracted from umbilical cord of full-term healthy neonates and stable cell lines were established through cell passage.2.The human umbilical cord mesenchymal stem cells were identified:(1)cell morphology was observed under inverted microscope;(2)alizarin red staining and oil red O staining were performed after osteogenesis and lipogenesis induction;(3)surface markers were detected by flow cytometry.3.HUC-MSC were divided into two groups:(1)untreated HUC-MSC(NativeMSC);(2)pretreated HUC-MSC(TNF-α&IFN-γ-MSC)were prepared by applying TNF-α and IFN-γ at a concentration of 10 ng/ml.4.Separate the exosomes from the supernatants of two groups of HUC-MSC using a high-speed centrifuge:(1)Untreated HUC-MSC derived exosomes(Native-Exo);(2)TNF-α and IFN-γ preprocessed HUC-MSC derived exosomes(TNF-α&IFN-γ-Exo).The morphology of exosomes was observed by transmission electron microscope and nanoparticle tracking analysis(NTA)analyze the diameter of exosomes and detect surface markers of exosomes using Western blot.Part 2: Protective effect of exosomes on kidney of DM mice1.T1 DM mice model was established by intraperitoneal injection of Streptozotocin(STZ)(60 mg/kg,5 days).The mice were divided into control group(NC group),diabetes group(DM group),diabetes +MSC-Exo intervention group(DM+Exo group),diabetes + pretreated MSC-Exo intervention group(DM+TNF-α&IFN-γ-Exo group).Five days after the last intraperitoneal injection of STZ,blood glucose levels were measured by tail vein to determine whether the DM mouse model was successfully established.After that,blood glucose and body weight of mice in each group were measured weekly.2.At the 12 th week after the successful establishment of T1 DM model,Native-Exo(50μg /mouse)in DM+Exo group and TNF-α&IFN-γ-Exo(50 μg /mouse)in DM+TNF-α&IFN-γ-Exo group were injected into tail vein twice a week for 5 weeks.The DM and NC groups were injected with PBS at the same amount.The glucose tolerance of mice in each group was measured 7 days after the last exosomes or PBS injection.At the18 th week of the experiment,the urine of mice was collected for urinary albumin/creatinine ratio(UACR),and then the body,kidney and spleen weight were measured,kidney coefficient and spleen coefficient were calculated to determine the effect of exosomes on physiological indexes of DM mice.3.To clarify whether exosomes can alleviate renal morphological changes in DM mice by HE,PAS,and Masson staining.4.The changes of CD4 +T cells and their subtypes in DM mice were detected by XII flow cytometry to determine whether exosomes could alleviate the overall inflammatory response of DM mice.5.The expression level of IL-6,IL-1β,TNF-α and IL-10 were measured by Western blot,the immunohistochemical staining of the above indicators was performed on the kidney of DM mice to evaluate whether exosomes can alleviate renal inflammatory damage in DM mice.6.To determine the effect of exosomes on monocytes in DM mice by flow cytometry.7.The proportion of macrophages with different phenotypes(M1,M2)in kidney of DM mice was measured by flow cytometry to determine whether exosomes could affect the changes of macrophage phenotype in kidney of DM mice.Part 3 Mechanism of exosomes regulating macrophage polarization1.Put RAW264.7 cell in the high sugar environment,the experiment was divided into control group(NC group,11.1 mmol/L glucose),high glucose group(HG group,35 mmol/L glucose),high glucose+MSC-Exo intervention group(HG+Exo group,35mmol/L glucose+10 μg/ml Exo),high sugar+pre-treatment MSC-Exo intervention group(HG+TNF-α&IFN-γ-Exo group,35 mmol/L glucose+10 μg/ml TNF-α&IFN-γ-Exo),where HG+ Exo group and HG+TNF-α&IFN-γ-Exo group,co-cultured with MSC-Exo and TNF-α&IFN-γ-Exo respectively,detect the expression of macrophage polarization marker iNOS(M1)and Arg1(M2)by Western blot to determine whether exosomes can affect changes in macrophage phenotype in high glucose environment.2.Using transcriptome sequencing method to detect MSC-Exo and TNF-α&IFN-γ-Exo regulates the changes in gene expression profile during macrophage polarization and identifies differential genes in high glucose environments.3.RT-qPCR and Western blot were used to identify the representative differential genes and screen the appropriate key differential gene.4.RAW264.7 cell were transiently transfected with ID3 overexpression plasmid,and Western blot was used to confirm the overexpression effect of ID3 overexpression plasmid.5.Apply empty vector plasmid and ID3 overexpression plasmid to transiently transfect RAW264.7 cultured in normal and high glucose environments respectively and divided into Vehicle group,ID3 group,Vehicle+HG group,and ID3+HG group.Then,Western blot was used to detect the expression levels of ID3,iNOS,and Arg1 in each group of cells,to clarify whether ID3 plays an important role in macrophage polarization under high glucose conditions.6.RAW264.7 cell were transiently transfected with empty vector plasmid and ID3 overexpression plasmid.The cells were divided into Vehicle group,ID3 group,ID3+Exo group and ID3 +TNF-α&IFN-γ-Exo group.Western blot was used to detect the expression level of iNOS and Arg1 in each group of cells to determine whether exosomes could affect the phenotype of macrophages under the condition of overexpression of ID3.7.RAW264.7 cell cultured in normal and high glucose environment were transiently transfected with empty vector plasmid and ID3 overexpression plasmid respectively.The cells were divided into Vehicle group,HG group,HG+Exo group,HG+TNF-α&IFN-γ-Exo group,HG+ID3+Exo group,HG+ID3+TNF-α&IFN-γ-Exo group.Western blot was used to detect the expression level of iNOS and Arg1 in each group of cells to determine whether the regulation of exosomes on macrophage polarization was realized by regulating the expression of ID3.Results:Part 1 Isolation and identification of exosomes from umbilical cord mesenchymal stem cells1.Analyzed under light microscopy,the cells grew stably on the wall and were in good condition after passage,suggesting that a stable passage cell line was established.2.(1)Analyzed under inverted microscope,cells grow stably and adhere to the wall in a long spindle shape;(2)After induction and staining,the cells showed multidirectional differentiation ability(3).Flow cytometry results showed positive expression of CD73,CD90 and CD105,while negative expression of CD34,CD45 and HLA-DR.The above results indicate that we have successfully obtained HUC-MSC.3.Two groups of HUC-MSC were obtained:(1)untreated HUC-MSC(NativeMSC);(2)pretreated HUC-MSC(TNF-α&IFN-γ-MSC).4.(1)Transmission electron microscopy showed that the shape of exosomes was round vesicle surrounded by double membrane,which was saucer-like structure;(2)NTA showed that the diameter of exosomes was mostly concentrated between 50 and200nm;(3)Western blot showed that CD9,CD63 and TSG101 were expressed at high level in exosomes but expressed at low level in mesenchymal stem cells.The results XIV above indicate that we have obtained exosomes which meet the criteria.Part 2: Protective effect of exosomes on kidney of DM mice1.After STZ injection,mouse with blood glucose levels ≥16.7 mmol/L on three consecutive days,STZ induced T1 DM mouse model was successful.Compared with NC group,DM model mice had significantly higher blood glucose level and slower body weight growth.2.MSC-Exo and TNF-α&IFN-γ-Exo can effectively reduce blood glucose levels in DM mice while increasing their weight gain rate,The effect of TNF-α&IFN-γ-Exo is more pronounced.MSC-Exo and TNF-α&IFN-γ-Exo can effectively alleviate glucose tolerance abnormalities in DM mice,but there is no significant difference in the effect between the two(P>0.05).MSC-Exo and TNF-α&IFN-γ-Exo can effectively reduce UACR in DM mice,the effect of TNF-α&IFN-γ-Exo is more significant.MSC-Exo and TNF-α&IFN-γ-Exo can alleviate the decrease in spleen coefficient in DM mice,but has no significant effect on slowing down the increase in kidney coefficient.Exosomes can improve some physiological indicators(blood glucose,body weight,glucose tolerance,spleen coefficient)in DM mice,and effectively alleviate kidney damage caused by DM.3.MSC-Exo and TNF-α&IFN-γ-Exo could alleviate the pathological changes such as inflammatory cell infiltration,glomerular basement membrane thickening and renal collagen deposition in diabetic mice,suggesting that exosomes could alleviate the renal injury caused by DM.4.MSC-Exo and TNF-α&IFN-γ-Exo could decrease the proportion of CD4 +T cells in spleen of DM mice,TNF-α&IFN-γ-Exo had better effect.MSC-Exo and TNF-α&IFN-γ-Exo could decrease the proportion of Th1 and Th17 cells in spleen of DM mice,and TNF-α&IFN-γ-Exo had more obvious effect.MSC-Exo and TNF-α&IFN-γ-Exo can increase the proportion of Th2 and Treg cells in spleen of DM mice,TNF-α&IFN-γ-Exo has better effect.These results suggested that exosomes could alleviate the inflammatory response in DM mice.5.MSC-Exo and TNF-α&IFN-γ-Exo can reduce IL-6,IL-1β,TNF-α and increase IL-10 in DM mice kidney,but The effect of TNF-α&IFN-γ-Exo is more significant.Exosomes can alleviate the inflammatory response in the kidneys of DM mice.6.MSC-Exo and TNF-α&IFN-γ-Exo can reduce the proportion of monocytes in the spleen of DM mice,but there is no significant difference in their effects(P>0.05),indicating that exosomes can reduce the recruitment level of monocytes in DM mice.7.MSC-Exo and TNF-α&IFN-γ-Exo can significantly inhibit M1 polarization of macrophages in the kidneys of DM mice,but there is no significant difference between the two(P>0.05).TNF-α&IFN-γ-Exo can significantly promote the polarization of macrophages in the kidneys of DM mice towards M2 type,but the effect of MSC-Exo is not significant.MSC-Exo and TNF-α&IFN-γ-Exo can reduce the ratio of M1/M2 macrophages in the kidneys of DM mice,the effect of TNF-α&IFN-γ-Exo is more pronounced.Prove that exosomes can inhibit the polarization of macrophages in the kidneys of DM mice towards M1 type,promote their polarization towards M2 type,and effectively reduce the ratio of M1/M2 macrophages,improving the inflammatory response in the kidneys of DM mice.Part 3 Mechanism of exosomes regulating macrophage polarization1.MSC-Exo and TNF-α&IFN-γ-Exo can decrease the expression of iNOS and increase the expression of Arg1 in RAW264.7 cell under high glucose environment,indicating that exosomes can promote macrophage polarization to M2 type and inhibit macrophage polarization to M1 type.2.Through comprehensive analysis of transcriptome sequencing results,we found that genes related to immune regulation,inflammatory response,and cell differentiation(CD5L,Parp16,Neurog2)were significantly upregulated.Genes related to transcriptional regulation,cell differentiation,and innate immunity(ID3,DMPK,Gbp7)were significantly downregulated.3.According to RT-qPCR and Western blot results,Inhibitor of DNA binding(ID3)may be a key differential gene in MSC-Exo and TNF-α&IFN-γ-Exo regulation of macrophage polarization in high glucose environment.4.Compared with the empty vector plasmid group and NC group,the ID3 overexpression plasmid can significantly increase the protein expression level of ID3 in RAW264.7 cell(P<0 0001),proving that the plasmid can be used for subsequent experiments.5.Compared with Vehicle+HG group and ID3+HG group,ID3 overexpression group had similar protein expression levels of ID3,iNOS and Arg1(P>0.05).The effect of ID3 overexpression on macrophage polarization markers was similar to that of high glucose,suggesting that ID3 plays an important role in macrophage polarization under high glucose.6.Compared with the ID3 overexpression group,the expression of iNOS was reduced in ID3+Exo and ID3+TNF-α&IFN-γ-Exo group,The expression of Arg1 was significantly increased in the ID3+TNF-α&IFN-γ-Exo group,but there was no significant difference in the expression level of Arg1 in the ID3+Exo group(P>0.05).These changes are related to MSC-Exo and TNF-α&IFN-γ-Exo under high glucose conditions has a similar effect on macrophage polarization marker proteins,suggesting that ID3 may be a key target for exosomes to regulate macrophage polarization.7.Compared with HG+Exo group and HG+TNF-α&IFN-γ-Exo group,the effect of inhibiting iNOS expression and increasing Arg1 expression in the HG+ID3+Exo group and HG+ID3+TNF-α&IFN-γ-Exo group was significantly weakened,indicating that exosomes may regulate macrophage polarization by inhibiting ID3 expression in high glucose conditions.Conclusion:1.MSC-Exo and TNF-α&IFN-γ-Exo could decrease the levels of blood glucose and urinary albumin/creatinine ratio(UACR)in DM mice,TNF-α&IFN-γ-Exo had better effect.2.MSC-Exo and TNF-α&IFN-γ-Exo could alleviate renal inflammatory response in DM mice,TNF-α&IFN-γ-Exo had better effect.3.CD4 +T cells,monocytes and macrophages play a role in MSC-Exo and TNF-α&IFN-γ-Exo regulating renal inflammatory response in diabetic mice.4.ID3 may be the key target of MSC-Exo and TNF-α&IFN-γ-Exo in regulating macrophage polarization under high glucose condition.MSC-Exo and TNF-α&IFN-γ-Exo may regulate the polarization of macrophages under high glucose by inhibiting the expression of ID3.