Effects and underlying mechanism of human adipose mesenchymal stem cells-derived exosomes on acute lung injury in septic mice
Objective To explore the effects and underlying mechanism of human adipose mesenchymal stem cells(ADSC)-derived exosomes on acute lung injury in septic mice.Methods The study was an experimental study.Human ADSC of passages 4-5 were selected,and exosomes in their supernatant were isolated and extracted by differential ultracentrifugation.Exosomes were then used after identification.Twenty-four adult male BALB/c mice were selected and divided into normal control group,simple cecal ligation and puncture(CLP)group,and CLP+ADSC-exosome group according to the random number table method(the grouping method was the same below),with 8 mice in each group.The mice in simple CLP group were injected with phosphate buffer after CLP surgery(to establish an animal model of acute lung injury in septic mice),the mice in CLP+ADSC-exosome group were treated according to the corresponding group name,and the mice in normal control group were only injected with phosphate buffer.At 24 hours after surgery,the morphology of lung tissue was observed by hematoxylin-eosin staining,the apoptosis of lung tissue cells was detected by in-situ end-labeling method,the content of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the serum of mice was detected by enzyme-linked immunosorbent assay,the content of malondialdehyde and superoxide dismutase(SOD)in lung tissue was detected by microplate reader,and the expressions of CD86 and CD206 in mouse lung tissue cells was detected by immunofluorescence method.Mouse macrophage RAW264.7 was taken and divided into blank control group,simple lipopolysaccharide(LPS)group,and LPS+ADSC-exosome group.The cells of LPS+ADSC-exosome group and simple LPS group were cultured by adding LPS+ADSC-exosome and LPS,respectively,and cells in blank control group were routinely cultured.Twelve hours after culture,the ATP content,the percentage of mitochondrial reactive oxygen species positive cells,as well as mitochondrial membrane potential in cells were detected by related detection kits.The mRNA expression levels of M1 polarization marker inducible nitric oxide synthase(iNOS),M2 polarization marker arginase-1(Arg1),and inflammatory factors TNF-α and IL-1β in cells were detected by real-time fluorescence quantitative reverse-transcription polymerase chain reaction method.Three samples were used for mRNA expression detection,and four samples were used for the detection of the other indicators.Results At 24 hours after surgery,the structure of mouse lung tissues in normal control group was clear and intact without inflammatory cell infiltration.Compared with that in normal control group,the lung tissue edema as well as the infiltration of inflammatory cells of mice was much more obvious in simple CLP group.However,compared with that in simple CLP group,the lung tissue edema of mice in CLP+ADSC-exosome group was significantly alleviated,the infiltration of inflammatory cells was significantly reduced,and the cell apoptosis and necrosis were significantly improved.Twenty-four hours after surgery,compared with that in normal control group,the levels of TNF-α and IL-1β in the serum of mice in simple CLP group were significantly increased(with t values of 50.82 and 30.81,respectively,P＜0.05);compared with that in simple CLP group,the levels of TNF-α and IL-1β in the serum of mice in CLP+ADSC-exosome group were significantly decreased(with t values of 16.36 and 19.25,respectively,P＜0.05).Compared with that in normal control group,the content of malondialdehyde in the lung tissue of mice in simple CLP group was significantly increased(t=9.89,P＜0.05);and the content of SOD was significantly decreased(t=5.01,P＜0.05);compared with that in simple CLP group,the content of malondialdehyde in the lung tissue of mice in CLP+ADSC-exosome group was significantly decreased(t=4.38,P＜0.05),and the content of SOD was significantly increased(t=2.97,P＜0.05).Twenty-four hours after surgery,compared with that in normal control group,the proportion of CD86 positive cells in the lung tissue of mice in simple CLP group was significantly increased,and the proportion of CD206 positive cells was significantly decreased;compared with that in simple CLP group,the proportion of CD86 positive cells in the lung tissue of mice in CLP+ADSC-exosome group was significantly decreased,and the proportion of CD206 positive cells was significantly increased.After 12 hours of culture,compared with that in blank control group,the ATP content of RAW264.7 cells in simple LPS group was significantly decreased(t=6.28,P＜0.05);compared with that in simple LPS group,the ATP content of RAW264.7 cells in LPS+ADSC-exosome group was significantly increased(t=4.01,P＜0.05).After 12 hours of culture,compared with(22±4)％in blank control group,(40±6)％of positive cells of mitochondrial reactive oxygen species in RAW264.7 cells in simple LPS group was significantly increased(t=5.04,P＜0.05);compared with that in LPS group,(30±5)％of positive cells of mitochondrial reactive oxygen species in RAW264.7 cells in LPS+ADSC-exosome group was signifiicantly decreased(t=2.65,P＜0.05).After 12 hours of culture,compared with that in blank control group,the mitochondrial membrane potential of RAW264.7 cells in simple LPS group was significantly decreased;the mitochondrial membrane potential of RAW264.7 cells in LPS+ADSC-exosome group was between those in blank control group and simple LPS group.After 12 hours of culture,compared with that in blank control group,the mRNA expressions of TNF-α,IL-1β,and iNOS in RAW264.7 cells in simple LPS group were significantly increased(with t values of 16.51,31.04,and 7.70,respectively,P＜0.05),and the decrease in the mRNA expression of Arg1 was not statistically significant(P＞0.05);compared with that in simple LPS group,the mRNA expressions of TNF-α,IL-1β,and iNOS in RAW264.7 cells in LPS+ADSC-exosome group were significantly decreased(with t values of 11.38,22.58,and 5.28,respectively,P＜0.05),and the mRNA expression of Arg1 was significantly increased(t=7.66,P＜0.05).Conclusions Human ADSC-exosomes may play a role in improving lung injury in septic mice by improving LPS-induced mitochondrial dysfunction in mice macrophages,inhibiting the polarization of macrophages toward M1,and reducing the inflammatory response.
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