Effect And Mechanism Of LIPUS Combined With Exosome-loaded Targeted Nanobubbles To Improve The Curative Efficacy Of Exosomes Derived From Adipose Mesenchymal Stem Cells In The Treatment Of Acute Myocardial Infarction

Part Ⅰ:Extraction and identification of adipose tissuederived mesenchymal stem cell（ADSCs）and exosomes derived from adipose tissue-derived mesenchymal stem cell（ADSCs）Objective: ADSCs were extracted from rat epididymal fat tissues,and ADSC-exo was extracted from cell culture supernatants.Methods:（1）ADSCs were extracted from SD rats by enzymatic digestion,and the morphology of cells was observed.Identification of ADSCs surface marker was detected by flow cytometry.The osteogenic and adipogenic differentiation abilities of the P3 generation ADSCs were also identified.（2）ADSC-exo was isolated from P3 generation ADSCs supernatant by ultracentrifugation.The morphology of the ADSC-exo was examined using transmission electron microscopy（TEM）.The size and distribution of the exosomes were measured using nanoparticle tracking analysis（NTA）.The expression of protein markers was detected by Western Blotting.Results:（1）The P3 generation of ADSCs exhibited a long,fusiform morphology and was arranged in a whirlpool-like shape.Flow cytometry analysis demonstrated that ADSCs were positive for mesenchymal stem cell surface markers CD29（99.82%）,CD44（99.77%）,and CD90（99.86%）and negative for CD34（2.89%）and CD45（2.23%）.The cell matrix exhibited red calcium deposition following the alizarin red staining and red fat drops following oil red staining.（2）The TEM results showed that ADSC-exo had a typical morphology with doublelayer membrane structure.The NTA results demonstrated that the size of the ADSCexo was distributed within a single peak with a medium size of 125.1 nm.Meanwhile,Western blotting revealed that the ADSC-exo was positive for specific exosome protein markers,such as CD9 and Tsg101,but negative for calnexin.Conclusion: We have successfully established stable ADSCs isolation and culture protocol,and obtained a huge number of high-purity ADSCs and ADSC-exo.Part Ⅱ:Effect of LIPUS on the release and uptake of ADSC-exo and the therapeutic efficacy in repairing acute myocardial infarctionObjective: To explore the effect of LIPUS on ADSC-exo release and uptake and its effect on the efficacy of ADSC-exo in repairing acute myocardial infarction.Methods:（1）The ADSCs in serum-free culture medium were treated with LIPUS at different intensities and durations to determine the optimal conditions for activating exosome release.The size distribution and concentration of ADSCs were measured by NTA.Apoptosis was evaluated using an annexin-V-FITC/PI kit and detected by flow cytometry.（2）Autophagy-related proteins p62 and LC3 were measured in ADSCs cultured in complete medium and FBS-free medium.After ADSCs were treated with autophagy inhibitor 3-MA,autophagy inducer rapamycin and LIPUS,the concentration of ADSCexo were detected by NTA,and the level of p62 and LC3 were detected by Western blotting.（3）The ADSCs in exosome-free culture medium were treated with LIPUS at different intensities and durations to determine the optimal conditions for activating exosome release.Apoptosis was evaluated using an annexin-V-FITC/PI kit and detected by flow cytometry.（4）After hypoxia-induced H9C2 cells were treated with LIPUS at different intensities and durations,the uptake of ADSC-exo was examined using confocal microscope analysis,and the apoptosis rate was detected by flow cytometry.（5）After acute myocardial infarction（AMI）rat models were constructed,the effect of LIPUS on the distribution of ADSC-exo in rats was observed by Optical Imaging system at multiple time points.At 72 h,the hearts were harvested for ex vivo imaging.The uptake of ADSC-exo in heart tissues were detected in frozen section,and the apoptosis rate was detected by TUNEL staining.（6）After AMI rat models were constructed,the rats were divided into five groups with different treatments 6 h after operation: sham group,PBS group,LIPUS group,EXO group,and EXO + LIPUS group.HE staining,Masson staining,TUNEL staining, cardiac function indicators of EF% and FS% were applied to evaluate treatment effect of acute myocardial infarction.Results:（1）As LIPUS irradiation at 0.6 W/m² for 20 or 30 min and 0.8 W/m² for 10,20,or 30 min induced similar effects on exosome release without changing the size distribution.The flow cytometry results revealed that LIPUS at 0.6 W/cm² for 30 min and 0.8 W/cm² for 20 or 30 min induced significant apoptosis compared with no LIPUS irradiation in serum-free culture.Interestingly,LIPUS at 0.6 W/cm² for 20 min and 0.8 W/cm² for 10 min did not cause significant apoptosis and can induce an increase in ADSC-exo production by approximately 50%.Therefore,0.8 W/cm² for 10 min was selected as the LIPUS irradiation parameter.（2）The ADSCs cultured in an FBS-free medium showed a decreased level of p62 and an increased level of LC3,suggesting autophagy was activated.The treatment with both 3-MA and LIPUS at 0.8 W/cm² for 10 min significantly decreased autophagy and the ratio of LC3-Ⅱ/LC3-I and increased the level of p62 and ADSC-exo release;in contrast,the rapamycin treatment had the opposite effect.Furthermore,LIPUS combined with 3-MA induced a higher level of p62 and exosome release and a lower ratio of LC3-Ⅱ/LC3-I compared with 3-MA or LIPUS alone.Moreover,LIPUS reversed the rapamycin-induced decrease in exosome release.（3）LIPUS irradiation at 0.8 W/cm² for 10 min increase ADSC-exo release by approximately 60% in a medium containing exosome-free FBS and did not cause significant apoptosis.（4）Confocal microscope analysis showed that hypoxia-induced H9C2 cells had taken up more exosomes when treated with LIPUS at 0.2 W/cm² for 10 min.The flow cytometry results illustrated that the combination of ADSC-exo and LIPUS can reduce apoptosis rate by nearly 50%.（5）Optical Imaging system and frozen section of heart tissue showed that LIPUS irradiation at 0.2 W/cm² for 20 min or 40 min did not promote the uptake of ADSC-exo in heart tissue,while LIPUS irradiation at 0.2 W/cm² for 40 min induced significant apoptosis compared with no LIPUS irradiation detected by flow cytometry.（6）HE staining,Masson staining,TUNEL staining and cardiac function showed that ADSC-exo could reduce cardiomyocytes fibrosis apoptosis,attenuate inflammation and infarct size,and improve EF% and FS%.However,LIPUS irradiation alone or combined with ADSC-exo did not increase the treatment efficacy.Conclusion: LIPUS can promote ADSC-exo release.Although LIPUS irradiation at 0.2 W/cm² for 10 min significantly promoted the uptake of ADSC-exo in hypoxiainduced H9C2 cells,LIPUS irradiation at 0.2 W/cm² for 20 min did not promote the uptake of ADSC-exo in heart tissue.The result indicated that LIPUS at 0.2 W/cm² for 20 min did not contribute to myocardial infarction repair.Part Ⅲ:SDF-1α high expressed ADSC-exo combined with exosome-loaded targeting nanobubbles in the treatment of AMIObjective: Treatment of AMI via local delivery of SDF-1α overexpressed ADSC-exo through CD81 antibody-conjugated nano-bubbles targeting infarcted myocardial tissue.Methods:（1）Blank nano-bubbles(NB^Blank)and CD81 antibody and cRGD-conjugated targeted nano-bubbles(TNB ^CD81-cRGD)targeting infarcted myocardial tissue were constructed.The size distribution and concentration of nano-bubbles were measured by NTA.The morphology of the nano-bubbles was examined using TEM.The nanobubbles were injected into the rats via the tail vein,and the hematological indexes and HE staining of the liver,kidney,spleen and lung were detected to evaluate the safety in vivo.（2）The binding of ADSC-exo to TNB^CD81-cRGD was detected by confocal microscopy in vitro.The distribution of NB^Blank and TNB^CD81-cRGD in AMI rats and the ability to carry exosomes to cardiac tissue were observed by Optical Imaging system,and frozen sections were used to observe the uptake of exosomes in heart tissue.（3）SDF-1α overexpressed lentivirus transfected P3 generation ADSCs,EXO^SDF-1α were isolated from ADSCs supernatant by ultracentrifugation.The morphology of the exosomes was examined using TEM.The size and distribution of the exosomes were measured using NTA.The expression of protein markers and SDF-1α was detected by western blotting.（4）The level of SDF-1α was detected by western blotting in PBS group,TNB group,EXO group,EXO + TNB group,EXO^SDF-1α group and EXO^SDF-1α + TNB group.（5）The expression of CXCR4 and CXCR7 on the surface of ADSCs of P1-3 generation was detected by flow cytometry.The effect of SDF-1α-CXCR4/CXCR7 axis on the migration of ADSCs was verified by Transwell chemotaxis assay.（6）The P1 generation of GFP-labeled ADSCs were injected into AMI rats via the tail vein.The homing of ADSCs was observed through frozen section in PBS group,TNB group,EXO group,EXO + TNB group,EXO^SDF-1α group and EXO^SDF-1α + TNB group.When CXCR4 and CXCR7 were blocked,the homing of ADSCs in con group,antiCXCR4 group,anti-CXCR7 group,and anti-CXCR4 + anti-CXCR7 group was also observed.（7）Rats were randomly divided into 7 groups: sham group,PBS group,TNB group,EXO group,EXO + TNB group,EXO^SDF-1α group and EXO^SDF-1α + TNB group.HE staining,ELISA and immunofluorescence staining of M1 macrophages（CD68+CD86+）,M2 macrophages（CD68+CD206+）,neutrophils（Ly6g+）to evaluate inflammatory response.Masson staining,TUNEL staining,cardiac function indicators of EF% and FS% were applied to evaluate treatment effect of myocardial infarction.（8）Rats were randomly divided into 3 groups: sham group,PBS group,EXO group.The relationship between behavioral indicators such as total distance,upright frequency,saccharine preference index and cardiac function indicators such as EF%,FS% and infarct size was studied.Results:（1）The size of NB^Blank and TNB^CD81-cRGD was approximately 350 nm,and a large number of hollow bubbles with a diameter less than 1 μm could be detected under the light microscope.The size of NB^Blank and TNB^CD81-cRGD was increased after 48 h,and the concentration was decreased after 24 h.No obvious hematological toxicity and liver,kidney,spleen and lung toxicity were observed in rats.（2）Confocal microscopy detected that a large amount of ADSC-exo or fluorescent secondary antibodies were bind to TNB^CD81-cRGD,which showed the successful construction of TNB^CD81-cRGD.Optical Imaging system and frozen section of heart tissue showed that the combination of LIPUS and TNB^CD81-cRGD could promote the uptake of ADSC-exo in heart tissue.（3）The NTA results demonstrated that the size of EXO and EXO^SDF-1α was mainly 30-150 nm.The TEM results showed that EXO and EXO^SDF-1α had a typical morphology with double-layer membrane structure.Meanwhile,Western blotting revealed that EXO and EXO^SDF-1α were positive for specific exosome protein markers,such as CD81 and Tsg101,furthermore,increased level of SDF-1α was detected in EXO^SDF-1α.（4）Western blotting results showed that the level of SDF-1α in heart tissues in EXO^SDF-1α group and EXO^SDF-1α + TNB group was significantly increased,and the level of SDF-1α in EXO^SDF-1α + TNB group was higher than that in EXO^SDF-1α group.（5）Flow cytometry analysis demonstrated that the expression of CXCR4 and CXCR7 on P1 generation ADSCs was 31.38% and 20.89%,respectively.However,the expression of CXCR4 and CXCR7 on P2-P3 generation was significantly decreased.Transwell assay confirmed that SDF-1α had a significant chemotactic effect on P1 generation ADSCs.When CXCR4 or CXCR7 was blocked,the cell migration was significantly reduced compared with the SDF-1α group.Furthermore,when both CXCR4 and CXCR7 were blocked,the cell migration was further reduced.These results demonstrated that the SDF-1α-CXCR4/CXCR7 axis was involved in mediating the migration of ADSCs in vitro.（6）The number of GFP+ cells in EXO^SDF-1α group and EXO^SDF-1α + TNB group were significantly higher than of the other 5 groups,and the number of GFP+ cells in EXO^SDF-1α + TNB group was significantly higher than that in EXO^SDF-1α group.When CXCR4 or CXCR7 was blocked,cell migration was significantly lower than that of SDF-1α group,and when both CXCR4 and CXCR7 were blocked,cell migration was significantly lower than that of single CXCR blocked.This result demonstrated that the SDF-1α-CXCR4/CXCR7 axis was involved in mediating the homing of ADSCs in vivo.（7）The level of IL-6 in rats treated with EXO^SDF-1α or EXO^SDF-1α + TNB was statistically lower than those treated with EXO,while IL-10 had the opposite trend.M1 macrophages and neutrophils were decreased in EXO^SDF-1α group and EXO^SDF-1α + TNB group.Moreover,M2 macrophages were increased in EXO^SDF-1α group and EXO^SDF-1α + TNB group compared to other groups.In EXO^SDF-1α group or EXO^SDF-1α + TNB group,increased number of arterioles,decreased number of TUNEL positive cells,reduced infarct size and improved EF% and FS% were observed compared to other AMI groups.Among them,the EXO^SDF-1α + TNB group had the best myocardial infarction repair efficiency.At 21 days after myocardial infarction,the myocardial infarction area was reduced by 35.5% compared with EXO treatment,the apoptosis in the peri-infarct area was reduced by 54%,and angiogenesis was increased by 101%,EF% increased by 8%,FS% increased by 6.2%.（8）Compared with sham group,total distance,upright frequency and saccharine preference index were decreased in AMI rats,indicating that AMI could induce depression symptoms.ADSC-exo treatment could relieve depression symptoms in AMI rats.Our analysis revealed that total distance,upright frequency and saccharine preference index were negatively correlated with infarct size,positively correlated with EF% and FS%。Conclusion:EXO^SDF-1α combined with TNB^CD81-cRGD can significantly promote exosomes uptake,promote endogenous stem cell homing,and greatly improve the therapeutic effect of AMI.Part Ⅳ:Mechanism of adipose-derived mesenchyma stem cells in relieving depression symptomsObjective: To examine the effects of ADSCs on depression symptoms and detected the changes in the composition of gut microbiota.Methods: CSDS mice were constructed,ADSCs or PBS were injected via the tail vein.Behavioral tests were used to evaluate depression-like behaviors,and 16 s RNA sequencing was used to detect changes in gut microbiota.Results:（1）CSDS mice showed obvious depression-like behavior in the social interaction test（SIT）,exercise（LMT）,tail suspension test（TST）and forced swimming test（FST）.ADSCs treatment via tail tein significantly reversed CSDS-induced depression behaviors.（2）The increase in family Lactobacillaceae,order Lactobacillales and decrease in order Micrococcales,order Rhizobiales,species Bacteroides acidifaciens,species Parasutterella sp.were observed in CSDS mice after ADSCs treatment.These changes may contribute to the antidepressant effects of ADSCs.Receiver operating characteristic（ROC）curves revealed that the family Lactobacillaceae and the order Rhizobiales are potentially important biomarker for the antidepressant effects of ADSCs in CSDS mice.Conclusion: ADSCs were effective in treating depression behaviors in CSDS model,which might be partly due to the regulation of abnormal composition of gut microbiota.Thus,ADSCs offer a promising therapeutic strategy for treating depression in patients.