The Mechanisms Of Mesenchymal Stem Cell-derived Exosome Mediated Inhibition Of Inflammation And Aging

Objective: Aging is a multifactorial biological process characterized by increased inflammatory response and the inability to adapt to changes in the environment.Aging is associated with systemic deterioration of multiple organs including the cardiovascular system.The prevalence of cardiovascular diseases is increased in aged people.The content of reactive oxygen increases during senescence.Therefore,the aim of this study is to investigate the effects of inflammation in aging and whether UMSC-Exo can inhibit inflammation and reduce aging via Lnc RNA MALAT1 in cardiomyocytes and aged mice.We cultured cardiomyocytes under H2O2 condition to mimic the oxidative stress environment in vivo and use aged mice or D-gal induced aged mice model to assess the impact of Lnc RNA MALAT1 of UMSC-Exo in inflammation and aging in cardiomyocytes and aged heart.Our research will pave the way to the development of new strategies to slow down aging and prevent aging associated heart diseases by inhibiting inflammation.Methods: H9C2 cell line was treated by H2O2 to mimic oxidative stress and aged mice were chosen as an in vivo model to study mechanism of inflammation and aging.1.To elucidate the senescent-like state of the H9C2 cells induced by oxidative stress,the H9C2 cells were exposed to H2O2,then the m RNA level of inflammation cytokine TNF-?and aging related gene p21 were examined by q RT-PCR and protein expression were analyzed by Western blot,and SA-?gal staining was also performed in this study;2.The NF-?B activity and the protein expression of p-p65 were detected to study the effects of oxidative stress,IKK? specific inhibitor IMD-0354,si IKK? and UMSC-Exo on NF-?B activation;3.In order to examine the effects of IMD-0354,si IKK?,UMSC-Exo in H2O2induced-H9C2 inflammation,aging and apoptosis,the H9C2 cells were pretreated with IMD-0354 or UMSC-Exo or transfected with si IKK? prior to H2O2 treatment and the expression of TNF-? and p21 were examined by q RT-PCR and Western Blot,and apoptosis was assessed by flow cytometry;4.The effect of UMSC-Exo on H9C2 cells proliferation was detected by Ed U proliferation kit;5.In order to study the impact ofUMSC-Exo in inflammation and aging in heart of aged mice,UMSC-Exo was injected in mice by caudal vein injection and the activity of NF-?B,the expression of TNF-? and p21 were examined;6.UMSC-Exo-si MALAT1 pretreated H9C2 cells or D-gal induced aging mice were used as in vitro and in vivo models to study the impact of Lnc RNA MALAT1 in UMSC-Exo in inflammation and aging in H9C2 cells and heart.Results: 1.Our data showed that both m RNA and protein levels of TNF-? and p21 are significantly increased in response to H2O2 treatment,and the number of SA-?gal-positive cells were also increased significantly,suggesting that oxidative stress can induce inflammation and aging in H9C2 cells;2.The NF-?B activity and the protein expression of p-p65 were increased in H9C2 cells treated with H2O2,which indicated that oxidative stress can activate NF-?B pathway,which can be inhibited by IMD-0354,UMSC-Exo and si IKK?;3.The expression of TNF-? and p21,and the level of apoptosis were inhibited in H9C2 cells by pretreatment with IMD-0354 or UMSC-Exo or by transfecting the cells with si IKK?,suggesting that IMD-0354,si IKK? or UMSC-Exo can inhibit oxidative stress induced inflammation,apoptosis,and reduce cell aging;4.UMSC-Exo pretreatment promoted the proliferation of H9C2 cells;5.The activity of NF-?B pathway and the expression of TNF-? and p21 were decreased significantly in the heart of aged mice,which indicated that UMSC-Exo can slow down heart senescence by inhibiting inflammation response;6.The inhibition of inflammation response and aging were significantly reduced by silencing the endogenous Lnc RNA MALAT1 in UMSC-Exo,which suggests that Lnc RNA MALAT1 in UMSC-Exo can inhibit inflammation response and slow down aging.Conclusions: UMSC-Exo can block the activity of NF-?B signaling pathway and inhibit inflammation and delay senescence through the action of Lnc RNA MALAT1;The binding of MALAT1 to the transcription factor NF-?B in nucleus inhibits the binding of transcription factor to TNF-? promoter,which reduces the expression of TNF-?.