The Effect And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cell Exudate On Delaying Diabetes Nephropathy By Activating Macrophages

Objective: To explore the therapeutic effect of human umbilical cord mesenchymal stem cells derived exosomes(huc MSC-Ex)on diabetes kidney diseases(DKD)in vivo and vitro,and to explore their cellular and molecular differential expression and expression of related signal pathways based on single cell RNA sequencing,so as to further understand the relationship between huc MSC-Ex and the pathogenesis of DKD.Methods: Extraction,identification,and concentration detection of extracellular vesicles: Human umbilical cord mesenchymal stem c-ells(huc MSCs)were isolated using umbilical cord tissue block atta-chment method,and extracellular vesicles were extracted from the supernatant of huc MSCs culture dish using ultracentrifugation metho-d;Protein blotting was used to detect surface positive markers such as CD63 and CD81 on huc MSC-Ex;Observe the morphology of huc MSC-Ex using transmission electron microscopy,and observe the particle size of the extracted exosomes by using nanoparticle trackin-g analysis technology;In vivo experiment,we verify that human u-mbilical cord mesenchymal stem cell derived exosomes(huc MSC-E-x)improve DKD mice: this experiment uses spontaneous diabetes db/db mice as the experimental group and db/m mice as the normal control group(NC).The experimental group is fed with high fat an-d high sugar,and the NC group is fed with common food for 10 weeks,and the fasting blood glucose(FBG)value is monitored wee-kly,regularly collecting urine samples to detect microalbumin in ur-ine.When the FBG value of db/db mice continues to be≥16.7mm-ol/L,the 24 h excretion of microalbumin in urine is significantly hi-gher than that of the NC group.The experimental group mice are more mentally depressed than the NC group with sparse and rough hair compared to the control group,more water,food,and excretion than the NC group,it can be considered that DKD modeling is suc-cessful;Randomly divide the mice with successful DKD modeling into DKD group(7 mice)and DKD+huc MSC-Ex group(7 mice),randomly select 7 db/m mice and record them as NC group.The DKD+huc MSC-Ex group received tail vein injection of huc MSC-Ex(10mg/kg)and the DKD group and NC group received tail vein inj-ection of the same dose of PBS for 8 weeks(3 times/week),meas-uring FBG and body weight(BW)every two weeks.After 8 week-s,the mice were euthanized,using a urine protein quantification ki-t to detect 24-hour urinary protein excretion rate in mice,Serum c-reatinine(Scr)and blood urea nitrogen(BUN)were measured using enzyme linkedimmunosorbent assay kit;Sc RNA-seq was performe-d on the dissociated kidney on one side,and hematoxylin eosin(H E)staining was used on the other side to assess the degree of path-ological changes in kidney tissue,immunohistochemistry was used to assess the proliferation of renal cells,immunofluorescence and flow cytometry were used to assess the distribution of related immun-e cells in kidney tissue.Useing sc RNA-seq to explore the change in the number of intrinsic cells in the kidney tissue before and afte-r huc MSC-Ex treatment and the potential molecular action pathway;taking the above DKD group and DKD+huc MSC-Ex group mice dissociated kidney tissue to prepare single cell suspension on the com-puter for sc RNA-seq,useing cluster dimension reduction method,small fiddle diagram,histogram,gene heat map and other bionomic to analyze the changes in the intrinsic cell community in the kidney before and after huc MSC-Ex treatment,combined with the results of kidney transcriptome chip,using information communication betw-een cell communities and differential gene enrichment analysis for personalized analysis and identification,utilizing the ligand receptor database to analyze intercellular communication relationships and to explore the proportion changes of intrinsic cells present in the kidney before and after huc MSC-Ex treatment,and infer their possible f-unctional relationships and related signaling pathways;In vitro expe-riments,we verified that huc MSC-Ex activated macrophages were t-he key to improve diabetes nephropathy: RAW264.7 cells were cult-ured in vitro,and huc MSC-Ex was added to the culture dish when the culture condition was good.The above cells were divided into RAW group and RAW+huc MSC Ex group.CCK8 experiment was performed to verify cell proliferation activity,and transwell experiment was performed to verify cell invasion ability,Flow cytometrywa-s was used to detect the expression of markers(F4/80+CD206+)of M2 in RAW+huc MSC-Ex group;Sc RNA-seq was used to analyze t-he proportion of renal proper cells before and after huc MSC-Ex an-d its possible signal transduction pathway,and coculture system of M2 and PT induced by high glucose was established.After 72 hour-s,the above CCK8 experiment and cell migration experiment were repeated;using quantitative real-time polymerase chain reaction(QRT-PCR)to verify the possible interaction pathway between M2 and PT induced by high glucose conditions.Results: Transmission electron microscopy observed huc MSC-Ex as a discshaped vesicle,and western blotting method found that huc MSC-Ex expressed specific CD81 and CD63 protein molecules;Nanoparticle Tracking Analysis(NTA)was used to track and analyz-e the extracellular vesicles,and we found that the diameter of the particles was mostly concentrated between 100-200 nm;Compared with the NC group,the FBG,BW,and 24-hour urinary albumin ex-cretion rates of the DKD group mice were significantly increased.Compared with the DKD group mice,the DKD+huc MSC-Ex group mice had a weight loss in FBG(P<0.05)and a decrease in 24-hour urinary albumin excretion rate(P<0.05).After 8 weeks of treatment,Scr decreased compared to the DKD group and had statistical signif-icance(P<0.05).BW and BUN decreased compared to the DKD g-roup,but there was no statistical significance(P>0.05);Renal pathology showed that compared with the NC group,HE staining in pa-thological tissue of the DKD group showed a significant increase in capillary bulb volume,expansion of the mesangialarea,and increased deposition of extracellular matrix;Although the glomerular volume of the DKD+huc MSC-Ex group cannot be reversed to normal compared to the NC group,it has significantly decreased compared to the DKD group with improved mesangial expansion and reduced extracellular matrix deposition;Immunohistochemistry showed that compared with the NC group,the positive rate of Ki67 in the DKD group was significantly reduced,indicating a decrease in renal ce-ll proliferation.Compared with the DKD group,the positive expres-sion of Ki67 in the DKD+huc MSC-Ex group was increased,indica-ting that after extracellular treatment,the proliferation of renal cells was increased;Sc RNA-seq analysis of kidney cells revealed that the-re were 10 groups of cell communities with difference after huc MSC-Ex treatment.Among them,the proportion of M2 and PT cells increased.The renal immunofluorescence results showed that the sur-face markers(F4/80+CD206+)of M2 cellsincreased after huc MSCEx treatment compared to the DKD and NC groups,indicating that huc MSC-Ex improves DKD by promoting the activation of M2 cell-s;RAW264.7 macrophages were cultured in vitro and divided into RAW group and RAW+huc MSC-Ex group.CCK8 experiment showed that the proliferation activity of cells in RAW+huc MSC-Ex group was significantly higher than that in RAW group(P<0.05)and huc MSC-Ex with a concentration of 10^8/ml was the best.Transwell experiment showed that the cell migration and invasion ability of RAW+huc MSC-Ex group was significantly higher than that of RAW group(P<0.001);The flow cytometry resultsshowed that the surface markers(F4/80+CD206+)of M2 cells increased significantly(P<0.05)after the addition of huc MSC-Ex compared to the DKD group,indi-cating that huc MSC-Ex can act on M2 cells.Combined with the in vivo immunofluorescence experimental results,it is more fully de-monstrated that huc MSC-Ex improves DKD by acting on M2 cells;TCMK-1 cells were cultured in vitro when they were in good grow-th condition,they were divided into TCMK-1+RAW group,TCMK-1+medium group,TCMK-1+huc MSC-Ex group,and TCMK-1+RAW+huc MS-C-Ex group.The RAW264.7 cell added to huc MSC-Ex wer-e cocultured with high glucose induced TCMK-1 cells,and then su-bjected to CCK8 experiment again.The results showed that compar-ed with other groups,the TCMK-1+RAW+huc MSC-Ex group showed an increase in cell proliferation while the 48 th hour with the ext-ension of time,The proliferation activity of its cells increased(P<0.05);The transwell experiment showed that compared with other gro-ups,cell migration in the TCMK-1+RAW+huc MSC-Ex group increa-sed significantly(P<0.001);Using sc RNA-seq to detect the express-ion level of molecules in macrophages,the results showed that Cd74 was significantly expressed in macrophages.Previous experimental results showed that extracellular vesicles can act on PT cells by activating macrophages.We plotted the interaction diagram of signal transduction pathways between M2 and PT cells after huc MSC-Ex s-timulation(Cirle diagram),and the results showed that the APP sig-nal transduction pathway is involved in the above process,Therefor-e,it is speculated that the APP/Cd74 sig-naling pathway may be a key pathway for huc MSC-Ex to delay DKD by activating macrophages on PT cells.The cell type dot plotwas drawn using sc RNA-se-q analysis,and the results showed that the APP/Cd74 signaling pat-hway was strongly expressed in M2 cell on PT cells.In vitro q RT-PCR experiments confirmed that the APP/Cd74 signaling pathway was expressed significantly in the DKD+huc MSC-Ex group compare-d to DKD group(P<0.001).Conclusion: Vivo experiments confirmed that huc MSC-Ex is the key component to protect renal function and delay the process of DKD;Sc RNA-seq,vivo and vitro experiments verify that M2 cells interact with PT cells to improve the process of DKD;Huc MSC-Ex may be involved in the process of activating M2 cells to improve the process of DKD by PT cells through APP/Cd74 signaling pathway.