| Chronic mountain sickness(CMS)occurs at a higher incidence in the plateau,which is characterized as one excessive erythrocytosis.It has been confirmed that the balance of cell proliferation and apoptosis of bone marrow hematopoietic cells is involved into excessive erythrocytosis of CMS.The PI3K-Akt pathway plays an important role in the process of erythropoiesis induced by erythropoietin(EPO)and vascular endothelial growth factor(VEGF).Activation of PI3K-Akt pathway can inhibit apoptosis by regulating apoptotic molecules such as Bcl-xl and caspase-9.The inhibitors of PI3K-Akt signal pathway play antitumor effects by regulating cellular apoptosis.CMS is a chronic benign proliferative disease of bone marrow erythroid cells caused by hypoxia.However,the mechanism remains unclear.The aim of the present study is to explore whether PI3K-Akt signaling pathway is involved in the pathogenesis of CMS.In Part I,the effect of PI3K-Akt signal pathway on apoptosis and proliferation of K562 cells was investigated.Akt1 was silenced by lentiviral-mediated sh RNA or overexpressed by Lentiviral Akt1 in the K562 cells cultured in 1% oxygen.And the proliferation and apoptosis of K562 cells were determined using cell counting kit 8 and flow cytometry,respectively.The protein levels of caspase-9 and Bcl-xl were measured by Western blotting.The results showed that the over-expression of Akt1 in K562 cells reduced the protein level of caspase-9(0.99 ± 0.059 vs 0.50 ± 0.09,P = 0.0127),increased the protein level of Bcl-xl(0.61 ± 0.08 vs 0.94 ± 0.02,P = 0.0134)and the cell proliferation(0.83 ± 0.053 vs 1.10 ± 0.071,P = 0.0367).But it did not significantly change the apoptosis of K562 cells(28.21 ± 8.45 vs 11.73 ± 0.72,P=0.1238)compared with that of the lentiviral control vector.Silencing of Akt1 in K562 cells resulted in a decrease in the protein level of Bcl-xl(1.0 ± 0.089 vs 0.54 ± 0.07,P = 0.0156)and an increase in the protein level of caspase-9(0.39 ± 0.11 vs 0.99 ± 0.069,P = 0.0097),and the cell proliferation was inhibited(0.83 ± 0.04 vs 0.59 ± 0.02,P = 0.074)and the cells apoptosis was promoted(25.42 ± 4.11 vs 58.90 ± 10.80,p = 0.0442),as compared with those of the lentiviral scramble vector.It is suggested that Akt1 affected the proliferation and apoptosis of K562 cells cultured in hypoxia through changed the expression levels of caspase-9 and Bcl-xl in part.In Part II,the effect of PI3K-Akt signal pathway on the apoptosis of erythroid progenitor cells in bone marrow of CMS patients was investigated in vitro.22 CMS patients and 20 non-CMS participants were involved in the present study.Bone marrow mononuclear cells(BMMNCs)were harvested,cultured and treated with Celecoxib and Perifosine in vitro for 72 hours.The apoptotic rate of erythroblasts,the m RNA expressions of Akt,Bcl-xl and caspase-9,and the protein levels of Akt,p-Akt,Bcl-xl and caspase-9 in erythroblasts were determined by flow cytometry(FCM),quantitative RT-PCR,and Western-blotting,respectively.And the effects of pretreatment by Akt1 inhibitors(Celecoxib and Prifosine)on apoptosis and expression of above molecules in the cultured erythroblasts were analyzed.The results showed that the apoptotic rate of cultured erythroblasts was lower in the CMS group than that in the non-CMS group(7.94 ± 3.01 vs 11.79 ± 4.94,P < 0.05).The m RNA expression of Akt1,the m RNA and protein expression of Bcl-xl were higher and the m RNA and protein expression of caspase-9 was lower in the CMS group than those in the non-CMS group(P < 0.05),respectively.After Perifosine intervention,the apoptosis rate of erythroblasts increased in both CMS group and non-CMS group(P < 0.001).And Perifosine induced a decrease in Bcl-xl m RNA and protein and p-Akt proteins and an increase in caspase-9 m RNA and protein in cultured erythroblasts(P < 0.05).In CMS patients,the hemoglobin concentration was negatively correlated with apoptotic rate(r =-0.5748,P = 0.0064)and positively correlated with Bcl-xl m RNA in erythroblasts(r = 0.6394,P = 0.0018),and the erythroblasts apoptotic rate was negatively correlated with the Akt1 m RNA(r =-0.5888,P = 0.0050)and Bcl-xl m RNA(r =-0.6942,p = 0.0005).These data demonstrated that the apoptosis was downregulated and the PI3K-Akt signaling pathway was activated in the bone marrow erythroblasts of CMS patients,and which can be reversed by the treatment of Akt1 inhibitor Perifosine.The PI3K-Akt signal pathway might be involved in the mechanism of decreased apoptosis of erythroblasts in CMS patients.In Part Ⅲ,the effect of PI3K-Akt signal pathway in pathogenesis of CMS and the effect of pretreatment by Akt inhibitors on it were further investigated in vivo using an animal model of CMS.100 healthy male Wistar rats from The Animal Experiment Center of Gansu College of Traditional Chinese Medicine were involved in this study.These 100 rats were randomly classified into six groups: normoxic group(normoxia and without Akt inhibitor,20 rats),hypoxia group(hypoxia but without Akt inhibitor,20 rats),Vehicle hypoxia group(hypoxia with vehicle 2m L/day,10 rats),low dose of celecoxib intervention group(hypoxia with celecoxib 20mg/kg/d,20 rats),high dose of celecoxib intervention group(hypoxia with celecoxib 40mg/kg/d,20 rats)and Perifosine intervention group(hypoxia with Perifosine 36mg/kg/d,10 rats).The rats of hypoxia group were fed in hypobaric oxygen chamber with simulation condition as the altitude of 5500 meters(pressure of 54.02 Kpa,10.8% oxygen concentration,22h/d),the rats of normoxic group were fed in Xining at the altitude of 2260 meters.After 30 days,the rats were sacrificed and the samples were harvested.The blood gas analysis and blood cell analysis were implemented using arterial blood,and the EPO level of plasma was detected by ELISA.The pulmonary artery pressure was measured in 5 rats of normoxic group and hypoxia group,respectively.The BMMNCs were isolated from bone marrow.The apoptotic rate and expressions of Akt1,Bcl-xl and caspase-9 proteins in CD71+CD45-erythroblasts were determined by FCM.The results showed that the pulmonary artery pressure was obviously higher in hypoxia group than that in normxia group(66.06 ± 17.36 vs 29.78 ± 4.47,P < 0.001).And the levels of HCT,HGB and RBC were also significantly higher in all hypoxia treatment groups than those in normoxia group(P < 0.05),respectively.However,there was no significant difference in the apoptotic rates of CD45-CD71+ erythrocytes and BMMNCs,and Akt1,Bcl-xl and caspase-9 proteins between the CMS and non-CMS rats(P > 0.05).And the Akt inhibitors treatment did not induce significant change in HCT,HGB,RBC,Akt,Bcl-xl and caspase-9 and the apoptotic rate of bone marrow erythrocytes in all hypoxia groups(P > 0.05).In conclusion,the present study demonstrateded that Akt1 regulates the proliferation and apoptosis of K562 cells in hypoxia through Bcl-xl and caspase-9.In CMS patients,erythroblasts apoptosis is downregulated and the PI3K-Akt signaling pathway is activated in bone marrow erythroblasts,which could be reversed by the treatment of Akt inhibitor.The PI3K-Akt signal pathway appeared to be involved in the mechanism of decreased erythroblasts apoptosis and accumulation of red blood cells in CMS.However,the effect of Akt inhibitor on the apoptosis of bone marrow erythroid progenitorswas not obvious in the CMS rat model.Therefore,it is necessary to perform further research to elucidate the mechanism of excessive accumulation of red blood cells and explore new therapeutic strategy in CMS. |
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