| Objective:To investigate the protective effect and mechanism of exosomes derived from bone marrow mesenchymal stem cells (BM-MSC) on Ischemia Reperfusion Injuries (IRI) in rats.Methods:(1) Rat-MSC were grown and isolated by adherent cell isolation. MSC surface molecular markers CD44, CD29 and CD34 were tested by flow cytometry. The MSC were added to osteoblast and lipocyte induction media. The multipotency of MSC is measured by observing the differentiation into osteoblasts and lipocytes. (2) Using Total Exosome Isolation Reagent and ultracentrifugation, exosomes were extracted from MSC and fibroblast (HFL-1) culture supernatants. The shapes and sizes of exosomes were characterized with a Transmission Electron Microscope and the expression of the surface molecular marker CD63 was tested by flow cytometry. (3) 30 male rats between 200g to 250g were selected. The selected rats were randomly grouped into the Sham-operated (sham) group, the Ischemia Reperfusion Injury (IR) group, the MSC-treated (IR+MSC) group, the MSC-derived exosome-treated (IR+MSC-ex) group, and the fibroblast-derived exosome-treated (IR+F-ex) group. The model for Ischemia Reperfusion Injury was constructed. The left renal pedicle was clamped shut for 45 minutes. Then the left renal artery was re-opened and the right kidney was removed at the same time. MSC or exosomes were injected through the renal artery when IR injury occurred. The IR group was given an equivalent amount of PBS instead. The sham group was given no treatment after re-opening the renal pedicle. (4) The rats were euthanized 48 hours after the operation. Whole blood samples, renal tissue samples fixed with formalin and frozen with liquid nitrogen were obtained. The serum creatinine and urea nitrogen levels were tested. Changes in the histomorphology of the renal tissue samples were examined by HE-stained tissue samples and the observed changes were used in the pathological evaluations with the Paller scoring criteria. Cellular apoptosis was examined by TUNEL staining. Protein and RNA were extracted from the frozen renal tissue samples. The expression levels of pro inflammatory cytokines IL-1β and TNF-α were examined by PCR. The expression levels of Caspase-3, Bcl-2, Bax, PI3K, P-Akt were examined by Western blotting.Results:(1) The characteristics of MSC:Rat bone marrow MSC showed long fusiform adherent growth. After passage, the cells showed high levels of proliferation and fusion, and were arranged in whirlpool or radial configurations. Flow cytometry determined that MSC expressed levels of CD29 and CD44 but low level of CD34. MSC successfully differentiated into osteoblasts and lipocytes after growing in osteoblast-and lipocyte-inducing media respectively. (2) The characteristics of exosome:Under the transmission electron microscope, the extracted particles had a microcapsular structure, were circular or oval-shaped with radii between 30-60nm and uniform in size. Flow cytometry determined the surface marker CD63 to be positive, confirming that the extracted particles were indeed exosomes. (3) On renal function: 48 hours after reperfusion, the MSC group and the MSC-ex group showed serum creatinine levels of (147.5±15.89,231.5±30.03 respectively) and urea nitrogen levels of (20.49±5.70,31.36±5.53 respectively). These levels are significantly lower compared to the serum creatinine and urea nitrogen levels of the IR group (482±36.87, 45.42±2.73). The MSC-ex group also showed statistically significant improvements over the F-ex group (442±41.98,46.89±3.21). (4) Comparison of the pathological changes of the kidney:HE-stained samples showed significantly lower pathological damage in groups MSC and MSC-ex, compared to the IR group. The Paller scores for groups MSC and MSC-ex (22.50±6.28,35.17±7.57 respectively) were much lower than that of the IR group (59.50±8.12) (P<0.001). The Paller score of the MSC-ex group (35.17±7.57) was also significantly lower than that of the F-ex group (58.67±8.59) (P<0.01). (5) Comparison of apoptosis:In the TUNEL staining test of apoptosis in renal tubular epithelial cells, the MSC and the MSC-ex group showed much lower positive cell counts (5.17±1.17,8.67±1.63 respectively) compared to the IR group(13.67±1.86) (P<0.001, P<0.01). The MSC-ex group also showed a much lower positive cell count (8.67±1.63) compared to the F-ex group (12.67±1.75) (P<0.01). (6) The changes of inflammatory factors:Real-time quantitative PCR analysis of the proinflammatory cytokines IL-1β and TNF-α showed that,48 hours after reperfusion, the proinflammatory cytokine levels in the IR group were significantly increased. Compared to the IR group, the MSC and MSC-ex groups showed similar levels of IL-1β (P>0.05) but significantly lower levels of TNF-α (P<0.01). Compared to the F-ex group, the MSC-ex group showed similar level of 1L-1β (P>0.05) but significantly lower level of TNF-α (P<0.05). (7) The changes of protein levels:In the Western blot test, the MSC and MSC-ex group showed reduced levels of Caspase-3 and Bax (P<0.05) but significantly increased levels of Bcl-2, PI3K and P-Akt (P<0.05), compared to the IR and F-ex group.Conclusion:(1) MSC-derived exosome helps protect against Ischemia Reperfusion Injuries in rats. (2) MSC-derived exosome protects the kidney by reducing inflammation and activating the PI3K-Akt pathway, which stimulates proliferation and suppresses apoptosis of renal tubular epithelial cells. |
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