Study On The Mechanism Of Adaptive Response In MAPK/P38 Induced By Low Dose Radiation In Vitro

Low dose radiation (LDR) can enhance the resistance of the DNA and chromosome damage induced subsequent high dose radiation, that is adaptive response (AR). To date, most of researches focused on the gene, protein and animal levels. However, the survival adptive response of normal somatic hematologic cells and its related gene regulation, signal transduction are unknown in domestic and abroad.In the present study, we separated and cultured human MSC in vivo, the 4h and 24h survival rate were observed after 2Gy high dose radiation, the 25mGy,75mGy and 200 mGy preirradiated at 41k 24h and 48h before 2Gy radiation. At last, we found the survival rate is most obviously 24h after 75mGy. We detected the apoptosis rate with confocal microscopy and FCM. The proteins expression of cyclinB1,cyclinD1,MDM2,Bcl-2 and Bax by immunohistochemistry assay and MAPK/P38 and its downstream proteins-ATF2 and other P53, P21 proteins with Western blot in both MSC and K562 cell lines. As control, the 75mGy and only high dose radiation were selected.The main results were as followed:1. The apoptosis rate were detected by confocal microscopy and FCM at 4h and 24h after high dose radiation that is subsequent after LDR. hence, the apoptosis of earlier and advanced stage is decreased in D1+24hD2 group (7.3±1.8) compared with D2 (16.2±3.3),D1+4h+D2 (8.8±1.5) and D1+48h+D2 (9.9±3.1) group, the difference shows significant deviation (P<0.05). but the the apoptosis of earlier and advanced stage of K562 in D1+24h+D2 group (11±2.8) shows no different compared with D2 (10.7±2.1,D1+4h+D2 (10.2±2.9) and D1+48h+D2 group.2. The expression of protein was detected with immunohistochemsitry method at 4h and 24h after D2:The CyclinB1 stain grade of MSC 4h after D2 in D1+4h+D2 (2.4±2.9),D1+24h+D2 (2.8±2.4) and D1+48h+D2 (2.2±1.4) group shows exceedingly decreased compared with D2 (5.4±3.9) group; such results was coincident with 24h, the CyclinB1 stain grade is each D1+4h+D2 (2.6±2.1),D1+24h+D2 (2.8±1.2),D1+48h+D2 (2.6±1.4) and D2 (5.8±2.4).The MDM2 stain grade of MSC 4h after D2 in D1+4h+D2 (5.2±1.9),D1+24h+D2 (5.4±1.4) and D1+48h+D2 (5.0±1.4) group shows exceedingly decreased compared with D2 (6.4±1.4) group; such results was coincident with 24h, the CyclinB1 stain grade is each D1+4h+D2 (5.2±2.9),D1+24h+D2 (5.4±2.4),D1+48h+D2 (5.2±1.4) and D2 (6.7±2.2).The CyclinB1,CyclinD1, MDM2 stain grade of K562 cell in D1+4h+D2,D1+24h+D2 and D1+48h+D2 shows no different deviations with D2 group.3. The expression of Bcl-2 was detected with immunohistochemistry method at 4h and 24h after D2:The Bcl-2 stain grade of MSC 4h after D2 in D1+24h+D2 (5.2±2.4) and D1+48h+D2 (4.2±1.8) group shows exceedingly decreased compared with D2 (0) group; the CyclinB1 stain grade is D1+48h+D2 (5.2±2.4) 24 h after D2, Howerver, other group expressed no protein. The Bcl-2 and Bax stain grade of K562 cell in D1+4h+D2,D1+24h+D2 and D1+48h+D2 shows no different deviations with D2 group.4. The expression of apoptosis-related proteins were detected by Western blot assay. The results showed that P53 and P21 decreased, but phospho-P38MAPK and ATF2 increased after D2 followed by LDR.The main conclusions were as followed:1. MSC but not K562 cell shows adaptive response induced by low dose radiation, the optimal dose and time fraction is 24h after 75mGy radiation.2. The expression of CyclinB1 decreased obviously at 4h and 24h after D2 compared D2 only.the mainly manifestation is G2 arrest. The decreased expression of MDM2 elucidated the AR has related to MDM2. However, the CyclinB1,CyclinD1,MDM2 in K562 cell showed no different deviation with D2 group, that provided light on the K562 has not adaptive response induced by LDR, and LDR did not induce cell cycle arrest.3. The expression of anti-apoptosis gene Bcl-2 elevated after low dose radiation, the gene showed related to adaptive response. MSC didn't express BAX protein in D2 group and D1+D2 group. the apoptosis and anti-apoptosis gene showed no change.4. The adaptive response has related to activing the MAPK/P38 signal pathway.Backing up all our dataes, we substantiated that MSC but not K562 has adaptive response induced by LDR, to utilize the LDR in clinical works, increase the effect of chemo-and radiao-therapy, decrease the side effect and serve clinical work further more. Blocking the P38MAPK pathway may be a novel target of drug supplements, it can be extrapolate to other tumor and diseases.