The Regulation Of Lysophophatidic Acid On Apoptosis And Autophagy And Their Relationship Study In Bone Marrow Mesenchymal Stem Cell

It has been demonstrated that stem cell transplantation can promote myocardial repair and regeneration in injury myocardium after myocardial infarction (MI), which has currently become a hot area of research. Because bone marrow mesenchymal stem cell (MSC) has many advantages that are not exist in other stem cells, they are considered to be an appealing sourse for cardiac engraftment in the treatment of myocardial infarction. MSCs play their therapeutic roles in myocardium repair mainly through differentiating into myocardial cells, promoting angiogenesis and paracrine role. However, MSCs transplanted into infarcted heart have a low survival rate for the cause of hypoxia/serum deprivation (H/SD) and increasing of reactive oxide species (ROS). Therefore, understanding the mechanisms of apoptosis and anti-apopotosis will provide an effective theoretical basis to improve the therapeutic effect of stem cell. Early investigation indicated that H/SD and hydrogen peroxide (H2O2) can induce MSCs apoptosis and further proved that lysophophatidic acid (LPA), as a kind of endogenous bioactive phospholipid signal molecules, could inhibit MSCs apoptosis induced by H/SD. However, whether LPA can play its anti-apoptosis role in the presence of hydrogen peroxide is not clear. Autophagy, a process for the degradation of protein aggregates and dysfunctional organelles, is required for cellular homeostasis and cell survival in response to stress and is implicated in endogenous protection. Emerging evidence shows that autophagy is associated with the protective effect of ischemic preconditioning and dual role in the situation of H2O2. Meanwhile, the role of LPA on autophagy and apoptosis and their relationship are not clear too.Therefore, hydrogen peroxide and hypoxia/serum deprivation are used to establish oxide stress and ischemic microenvironments models to study the apoptosis and autophagy effects induced by LPA in rat bone marrow mesenchymal stem cells. We further confirm whether the regulation of autophagy by LPA can influence its regulation of apoptosis. Our main work includes two aspects as follows:Part Ⅰ:The regulation of LPA on hydrogen oxygen induced bone marrow mesenchymal stem cell apoptosis and its mechanism study(1) H2O2-induced BMSCs apoptosis presents dose and time dependent. Cells treated with different concentrations of H2O2(50,100,150,200,250μM) for4hours revealed that apoptosis characteristics became significant difference from150μM H2O2and reached top at250μM H2O2. Comparing to the control group, cells in H2O2-treated group presented an abnormal morphology with shrinkage in cell size, chromatin condensation and typical fragmented nuclei. Different period experiment (0,2,4,6,8h) was carried on and from the fourth hour cleaved-caspase3protein level begun to increase obviously. Conclusion together, this data suggests that H2O2induced apoptosis dependently on dose and time and the apoptosis and autophagy are began to be markedly induced in250μM H2O2for4h.(2) LPA can effectively inhibit BMSCs apoptosis induced by H2O2BMSCs were exposed to increasing concentrations of LPA (1,5,10,25,50μM) and followed by exposure to250μM H2O2for4hours. After staining of hoechst.33342, control cells showed normal elongated stem cell morphology with large regular nuclei, but cells treated by H2O2appeared shrunken cell size and fragmented nuclei. When LPA mixed, apoptosis cells decreased pronouncedly closing to normal group. Flow cytometry analysis indicated that exposure of BMSCs to250μM H2O2resulted in apoptosis percentage of about35%Annexin V+/PI-cells (early stage of apoptosis) and5%Annexin V+/PI+cells (middle/late stage of apoptosis). Western blotting results showed that cleaved-caspase3, a key marker of apoptosis, was remarkably inhibited by LPA comparing to H2O2treatment group. These data demonstrated that10μM LPA could effectively inhibit apoptosis induced by250μM H2O2in rat BMSCs for4h.(3) LPA inhibits H2O2-induced BMSCs apoptosis through LPAR-3/Gi-coupled pathway.①BMSCs were pre-treated with antagonist of LPAR-1/3receptor Ki16425(10μM) for90min and Gi protein inhibitor PTX(200ng/ml) forl6h before the exposure to H2O2and LPA. Western blot analyses showed that Ki16425and PTX could significantly increase cleaved-caspase3protein level compared to LPA treatment group. Flow cytometric analyses revealed that Ki16425and PTX could markedly raise the percentage of Annexin V+/PI" cells in the presence of LPA.②Lparl and Lpar3were separately knocked down by siRNA transfection and we found that cleaved-caspase3protein level increased markedly after LPAR-3-siRNA mixing, but not by the addition of LPAR-1-siRNA.③Gi2and Gi3were also knocked down by Gi2-siRNA and Gi3-siRNA respectively and the result showed that cleaved-caspase3protein level both increased after transfection of Gi2-siRNA and Gi3-siRNA.④Western blotting showed that LPA could elicit the phosphorylation of ERK1/2and AKT with a significance increase in the5min and10 min, respectively after stimulation of LPA in rat BMSCs, which could be canceled in the presence of Ki16425and PTX.⑤The ERK1/2inhibitor U0126and PI3K inhibitor LY294002both increased cleaved-caspase3expression and attended the LPA’s anti-apoptosis. Flow cytometric analyses appeared that Uo126and LY294002both took part in anti-apoptosis role in early stage of apoptosis (Annexin V+/PI-). These data indicate that LPA inhibits H2O2-induced apoptosis mainly through Gi coupled Lpar3receptor to activiat ERK1/2and PI3K/AKT pathways to inhibition transmissionis of apoptosis signal.(4) Autophagy showed a protective effect in H2O2-induced BMSCs apoptosis.①It iwas showed that LC3Ⅱ/I ratio increased significantly comparing to control from the forth hour and the expression of P62showed a decease tendency as time went on. However, the expression of BECN has no significant difference during these groups.②autophagy promoter GF109203x and inhibitor Bafilomycin Al were separately pre-added in cell culture medium for90min and followed by exposure to H2O2for4h. Western blotting result indicated that GF109203x partly decreased the expression of cleaved-caspase3. But Bafilomycin A1had nothing role in BMSCs survival or apoptosis. These data explained that H2O2-induced autophagy showed a protective role in BMSCs’survival.(5) LPA protects BMSCs from HOO2-induced apoptosis independent of autophagy.①BMSCs were exposed to increasing concentrations of LPA (1,5,10,25,50μM) and10μMLPA could effectively increase the transformation rate of LC3B I to LC3BⅡ but with no further decrease of P62expression. However, the expression of Beclinl had no significant differences among these groups.②Bafilomycin A1, a autophagy flux inhibitor aiming at the fusion of autophagosome and lysosome, was used to interrupted most macroautophagy pathway. Comparing with LPA+H2O2group, LPA+H2O2+Bafilomycin Al group showed obvious anti-apoptosis role with no significant difference between them. Accordingly, we concluded that LPA rescued BMSCs from H2O2-induced apoptosis was independent of autophagy.Part I investigation demonstrated that:H2O2induced BMSCs apoptosis in time-and dose-dependent ways; LPA could effectively inhibit H2O2induced apoptosis; LPA inhibited H2O2-induced apoptosis via Gi-coupled LPAR-3receptor to activate ERK1/2and PI3K/AKT signaling pathway; LPA could promote H2O2-induced autophagy which has no connection with the anti-apoptosis role of LPA.Part II:The regulation of autophagy in the process of H/SD-induced bone marrow mesenchymal stem cell apoptosis influenced by lysophophatidic acid.(1) Autophagy showed a protective effect in H/SD-induced BMSCs apoptosis.①It iwas showed that LC3Ⅱ/Ⅰ ratio increased significantly comparing to control from the sixth hour and the expression of P62showed a decease tendency as time went on. However, the expression of BECN has no significant difference during these groups.②autophagy promoter GF109203x and rapamycin and autophagy inhibitor Bafilomycin A1and3-MA were separately pre-added in cell culture medium. Western blotting result indicated that GF109203x and rapamycin partly weakened the expression of cleaved-caspase3and increased the ratio of Bcl2/Bax. But Bafilomycin Al and3-MA increased the expression of cleaved-caspase3and decreased the ratio of Bcl2/Bax. These data explained that H/SD-induced autophagy had a protective role in BMSCs survival.(2) The autophagy induced by LPA in H/SD situation taked part in the anti-apoptosis process of LPA.①Different concentrations of LPA (1,5,10,25,50μM) could increase the transformation rate of LC3B I to LC3B II but with no further decrease of P62expression. However, the expression of Beclinl had no significant differences among these groups.②LPA could effectively inhibit cleaved-caspase3level and increase the ratio of Bcl2/Bax.③aotophagy inhibitor Bafilomycin Al and3-MA weakened the anti-apoptosis role of LPA comparing with LPA group. These data suggested that the dcrease of anti-apoptosis role of LPA has a relationship with the inhibition of autophagy.(3) LPA promoted MSCs autophagy mainly through Gi coupled and other G protein coupled Lpar3receptor pathways.①Lparl/3inhibitor Ki16425could evidently prevent the pro-autophagy role of LPA.②Gi protein inhibitor PTX could partly suppress autophagy induced by LPA in H/SD.(4) Autophagy induced by LPA in the situation of H/SD dependented on the pathways of ERK1/2and AMPK.①Our group has demonstrated that LPA can stimulate phosphorylation of ERK1/2and AKT through Gi coupled Lparl/3pathway. We further proved that LPA could activate AMPK’s phosphorylation which could be inhibited by Lparl/3inhibitor Kil6425but with no influence with the adding of PTX.②PI3K inhibitor LY294002, ERK1/2inhibitor U0126and AMPK inhibitor Compound C could all attenuate the anti-apoptosis of LPA.③Only U0126and Compound C could prevent the pro-autophagy effect of LPA. These results suggested that ERK1/2and AMPK took part in LPA-induced autophagy.PartⅡinvestigated that H/SD could induce MCSs apoptosis and autophagy in time dependent way and from sixth hour the level of apoptosis and autophagy began to increase markedly. H/SD-induced autophagy in MSCs showed a protective role in the cel survival. LPA could promote MSCs autophagy which was proved to take part in the anti-apoptosis reponse against H/SD. It was finaly testified that LPA-elicited autophagy mainly by binding Lparl/3coupling Gi protein to activate ERK1/2pathway and other G protein to activate AMPK pathway.In this study it was proved first that H2O2-induced apoptosis could be cancled by LPA. We further found that LPA played its anti-apoptois role against H2O2mainly by bingding Lpar3receptor coupling Gi protein to activate PI3K/AKT and ERK1/2pathways, along with the anti-apoptosis role of LPA against H/SD, which revealed the important values of LPA in the stem cell engrafment for the treatment of yocardial infarction. Besides, we first analyzed the pro-autophagy response of LPA under the condtions of hydrogen peroxide and H/SD. Although autophagy induced by LPA under the existence of H2O2had no connection with LPA’s anti-apoptosis role, LPA-induced autophagy in H/SD showed a significant role for its anti-apoptosis effect. The pro-autophagy mechanism by LPA was mainly through Lparl/3coupling Gi protein to activate ERK1/2pathway and other G protein to activate AMPK pathway, which provides a new sight for improving the effect of stem cell transplanting in the treatment of myocardial infartion. Given that autophagy involves in a variety of cell biology processes such as cell proliferation, differentiation and migration, further in-depth studies for the relationships between the autophagy regulation of LPA and variety of cytological behaviors will provide a strong evidence for the stem cell engrafment for the treatment of myocardial infarion.