International Journal of
Molecular Sciences Article
p38 MAPK Inhibitor Insufficiently Attenuates HSC Senescence Administered Long-Term after 6 Gy Total Body Irradiation in Mice Lu Lu 1 , Yue-Ying Wang 1 , Jun-Ling Zhang 1 , De-Guan Li 1, * and Ai-Min Meng 1,2, * 1
2
*
Institute of Radiation Medicine, Chinese Academy of Medical Science and Peking Union Medical Collage, Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Tianjin 300192, China;
[email protected] (L.L.);
[email protected] (Y.-Y.W.);
[email protected] (J.-L.Z.) Institute of Laboratory Animal Science, Chinese Academy of Medical Science and Peking Union Medical Collage, Beijing 100021, China Correspondence:
[email protected] (D.-G.L.);
[email protected] (A.-M.M.); Tel.: +86-22-8568-2353 (D.-G.L.); +86-10-6777-6299 (A.-M.M.); Fax: +86-22-8568-3033 (D.-G.L.)
Academic Editor: Atsushi Matsuzawa Received: 27 April 2016; Accepted: 3 June 2016; Published: 8 June 2016
Abstract: Senescent hematopoietic stem cells (HSCs) accumulate with age and exposure to stress, such as total-body irradiation (TBI), which may cause long-term myelosuppression in the clinic. However, the methods available for long-term myelosuppression remain limited. Previous studies have demonstrated that sustained p38 mitogen-activated protein kinases (p38 MAPK) activation in HSCs following exposure to TBI in mice and the administration of its inhibitor twenty-four hours after TBI may partially prevent long-term myelosuppression. However, long-term myelosuppression is latent and identified long after the administration of radiation. In this study, we investigated the effects of SB203580 (a small molecule inhibitor of p38 MAPK) on long-term myelosuppression induced by TBI. Mice with hematopoietic injury were injected intraperitoneally with SB203580 every other day five times beginning 70 days after 6 Gy of 137 Cs γ ray TBI. Our results at 80 days demonstrated that SB203580 did not significantly improve the TBI-induced long-term reduction of peripheral blood cell and bone marrow nucleated cell (BMNC) counts, or defects in hematopoietic progenitor cells (HPCs) and HSC clonogenic function. SB203580 reduced reactive oxygen species (ROS) production and p-p38 expression; however, SB203580 had no effect on p16 expression in the HSCs of mice. In conclusion, these findings suggest that treatment with SB203580 70 days after TBI in mice inhibits the ROS-p38 oxidative stress pathway; however, it has no therapeutic effect on long-term myelosuppression induced by TBI. Keywords: p38; ionizing radiation; bone marrow; long-term myelosuppression
1. Introduction Senescent cells accumulate with aging and multiple physiological and pathological processes. Cellular senescence is multifunctional, including protection against cancer and participation in complex biological processes, such as embryonic development [1], tissue repair [2], aging and age-related disorders [3]. Therefore, effective therapeutic strategies for the treatment of age-related diseases and improvements in healthy lifespans are needed. Cell senescence primarily occurs via two signaling pathways, including the p53-p21 pathway, which is activated by DNA damage or shortened telomeres, and the p16-Rb pathway, which is activated by the p38 MAPK cascade. The activation of these pathways may induce senescence, and the p38-p16 pathway plays an important role in the senescence pathway [4,5]. Our previous study demonstrated that hematopoietic stem cells (HSCs) undergo senescence in vitro and in vivo Int. J. Mol. Sci. 2016, 17, 905; doi:10.3390/ijms17060905
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following exposure to ionizing radiation (IR) [6,7]. The senescent HSCs induced by IR expressed increased senescence-associated-β-galactosidase (SA-β-gal) activity and p16 levels. In addition, the p38 expression and ROS levels increased [8]. Increasing evidence indicates that the p38-p16 pathway plays an important role in the regulation of HSCInt. self-renewal and J. Mol. Sci. 2016, 17, 905 the remission of hematopoietic cell senescence induced by 2IR of 9 in vitro and in vivo [8,9]. The inhibition of p38 activation with the small molecule inhibitor SB203580 in vivo following exposure to ionizing radiation (IR) [6,7]. The senescent HSCs induced by IR promotes ex vivo HSC expansion [10]. Moreover, the inhibition of p38 MAPK with SB203580 24 expressed increased senescence-associated-β-galactosidase (SA-β-gal) activity and p16 levels. In h after TBI attenuates residual BMincreased damage[8]. [9]. Furthermore, chemical inhibition of p38 addition, the p38 IR-induced expression and ROS levels rejuvenates Increasing aged satellite cells indicates and promotes following injuryrole [11].in In evidence that themuscle p38-p16regeneration pathway plays an important thecontrast of HSC self-renewal andmyelosuppression the remission of hematopoietic cell senescence induced and by IRexhibits to acuteregulation myelosuppression, long-term is latent [7,12], is long-lasting, in vitro and vivo [8,9]. The inhibition of p38 we activation with the small molecule inhibitor SB203580 little tendency forin recovery [13]. Therefore, investigated whether administration of the p38 promotes ex vivo HSC expansion [10]. Moreover, the inhibition of p38 MAPK with SB203580 24 h inhibitor SB203580 after ionizing radiation-induced HSC senescence may ameliorate TBI-induced after TBI attenuates IR-induced residual BM damage [9]. Furthermore, chemical inhibition of p38 long-term myelosuppression. rejuvenates aged satellite cells and promotes muscle regeneration following injury [11]. In contrast to acute myelosuppression, long-term myelosuppression is latent [7,12], is long-lasting, and exhibits 2. Results little tendency for recovery [13]. Therefore, we investigated whether administration of the p38 inhibitor SB203580 after ionizing radiation-induced HSC senescence may ameliorate TBI-induced 2.1. Effects of SB203580 on Peripheral Blood Cells after TBI long-term myelosuppression.
Previous studies have demonstrated that exposure to sub-lethal doses of 137 Cs γ ray TBI led 2. Results to long-term BM suppression and induced a decrease in the peripheral blood cell counts [14,15]. To investigate theofSB203580 treatment on after IR-induced hematopoietic system injury, the numbers 2.1. Effects SB203580 on Peripheraleffects Blood Cells TBI of peripheralPrevious blood cells were analyzed 80 days after 6 Gy TBI. Mice irradiated with 6 Gy137 Cs γ rays studies have demonstrated that exposure to sub-lethal doses of 137Cs γ ray TBI led to receivedlong-term injections SB203580and or vehicle theperipheral schedules (Figure 1A). [14,15]. The numbers of BMofsuppression induced according a decrease intothe blood cell counts To WBCs, RBCs, HGB, platelets in the effects vehicle-treated andhematopoietic SB203580-treated exhibited substantial investigate theand SB203580 treatment on IR-induced systemmice injury, the numbers of peripheral were 80 days afterthe 6 Gy TBI. Mice irradiated with 6the Gy137 Cs γ rays of the reductions 80 daysblood aftercells 6 Gy TBIanalyzed compared with control mice; however, treatment received injections of SB203580 oreffect vehicle to the (FigureHGB, 1A). or Theplatelets numberscompared of irradiated mice with SB203580 had no onaccording the number ofschedules WBCs, RBCs, WBCs, RBCs, HGB, and platelets in the vehicle-treated and SB203580-treated mice exhibited with the vehicle-treated mice (Figure 1B). These findings suggest that SB203580 treatment 70 days after substantial reductions 80 days after 6 Gy TBI compared with the control mice; however, the 6 Gy TBItreatment does not hematopoietic system of ameliorate the irradiatedTBI-induced mice with SB203580 had no effect on the injury. number of WBCs, RBCs, HGB, or platelets compared with the vehicle-treated mice (Figure 1B). These findings suggest that SB203580
2.2. Effects of SB203580 BMNC Counts CFU-GMTBI-induced after TBI hematopoietic system injury. treatment 70 dayson after 6 Gy TBI does and not ameliorate
To investigate the effects of SB203580 treatment on TBI-induced long-term BM injury, we initially 2.2. Effects of SB203580 on BMNC Counts and CFU-GM after TBI analyzed the (bone marrow nucleated cell) BMNC counts 80 days after 6 Gy TBI. The number of BMNC To investigate the effects of SB203580 treatment on TBI-induced long-term BM injury, we in the vehicle-treated and SB203580-treated mice was decreased compared with the control mice. initially analyzed the (bone marrow nucleated cell) BMNC counts 80 days after 6 Gy TBI. The However, the difference between the mice that received SB203580 and vehicle was not statistically number of BMNC in the vehicle-treated and SB203580-treated mice was decreased compared with significant (Figure 2A). Furthermore, we performed CFC assay to determine whether SB203580 the control mice. However, the difference between theamice that received SB203580 and vehicle was treatment thesignificant colony-forming capacity of HPCs we from the irradiated exposure notincreased statistically (Figure 2A). Furthermore, performed a CFC mice. assay Radiation to determine whether SB203580 treatment increased the colony-forming HPCs the irradiated significantly reduced the frequencies of CFU-GM 80 dayscapacity after 6ofGy TBIfrom (Figure 2B). However, mice. exposure significantly reduced the frequencies of CFU-GM days after 6 Gy TBI there were noRadiation significant differences in the CFU-GM frequencies between80the SB203580-treated and (Figure 2B). However, there were no significant differences in the CFU-GM frequencies between the vehicle-treated mice. These findings suggest that SB203580 has no effects on the BMNC numbers or SB203580-treated and vehicle-treated mice. These findings suggest that SB203580 has no effects on colony formation of CFU-GM 70 days after of 6 Gy TBI. 70 days after 6 Gy TBI. the BMNC numbers or colony formation CFU-GM
Figure 1. Cont.
Figure 1. Cont.
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Figure Effects SB203580on onTBI-induced TBI-induced myelosuppression. myelosuppression. (A) or or Figure 1. 1. Effects ofof SB203580 (A)Mice Micewere weresham-irradiated sham-irradiated Figure 1. Effects on TBI-induced myelosuppression. (A)SB203580 Mice were sham-irradiated or irradiated with 6 of GySB203580 TBI; 70 days after TBI, they were treated with or vehicle every other irradiated with 6 Gy TBI; 70 days after TBI, they were treated with SB203580 or vehicle every other irradiated with 6 Gy 70 days aftermice TBI,were they sham-irradiated were treated with SB203580 vehicle every other day for five times as TBI; shown. Control and receivedorinjections of vehicle; day forfor five times asasshown. Control mice were sham-irradiated andreceived receivedinjections injections of vehicle; day five times shown. Control mice were sham-irradiated and of vehicle; (B) Peripheral blood cells were counted 80 days after 6 Gy TBI. The number of white blood cells (B)(B) Peripheral blood cells were counted 80 days afterafter 6 Gy6TBI. of white blood blood cells (WBCs), Peripheral blood cells were counted 80 (HGB), days Gy The TBI.number The number of white cells (WBCs), red blood cells (RBCs), hemoglobin and platelets (PLT) in the peripheral blood were red(WBCs), blood cells (RBCs), hemoglobin (HGB), and platelets (PLT) in the peripheral blood were quantified red blood cells (RBCs), hemoglobin (HGB), and platelets (PLT) in the peripheral blood were quantified 80 days after 6 Gy TBI. The data are presented as the means ± SD. n = 12 mice/group. a p < 0.05 80 quantified days after 6 Gy TBI. The data areThe presented the means n = 12 mice/group. days after 6 Gy TBI. data areaspresented as ˘ theSD. means ± SD. n = 12 mice/group. a p < 0.05 vs.80 control. vs.acontrol. p < 0.05 vs. control.
Figure 2. Effects of SB203580 on TBI-induced BMNC counts and CFU-GM. Mice were treated with Figure 2. of SB203580 on TBI-induced BMNC counts and CFU-GM. Mice were treated with vehicle orEffects SB203580 70 dayson after exposure toBMNC 6 Gy TBI, as described in the experimental section. Figure 2. Effects of SB203580 TBI-induced counts and CFU-GM. Mice were treated with vehicle or SB203580 70 days after exposure to 6 Gy TBI, as described in the experimental section. (A) The number of70 BMNCs was counted euthanized 80 days after 6 Gysection. TBI; vehicle or SB203580 days after exposureafter to 6the Gymice TBI, were as described in the experimental (A) The The number of BMNCs wasHPCs counted after the was micemeasured were euthanized 80 days Gy TBI; clonogenic function in BMNCs via 80 a days CFC assay.after The6TBI; data (A)(B) The number of BMNCs was of counted after the mice were euthanized after 6 Gy (B)are The (B) The clonogenic function of HPCs in BMNCs was measured via a CFC assay. The data are a presented as the means ± SD. n = 12 mice/group; p < 0.05 vs. control. clonogenic function of HPCs in BMNCs was measured via a CFC assay. The data are presented as the presented as the means ± SD. n = 12 mice/group; a p < 0.05 vs. control. means ˘ SD. n = 12 mice/group; a p < 0.05 vs. control.
2.3. Effects of SB203580 on TBI-Induced Long-Term HSC Injury 2.3. Effects of SB203580 on TBI-Induced Long-Term HSC Injury 2.3. Effects SB203580 oninvestigated TBI-Inducedthe Long-Term Injurytreatment on TBI-induced long-term HSC We of subsequently effects of HSC SB203580 We subsequently investigated the effects of SB203580 on TBI-induced long-term HSC injury. First, we analyzed the HSC clonogenic function viatreatment a cobblestone area-forming cell (CAFC) We subsequently investigated the effects offunction SB203580 treatment on TBI-induced long-term HSC injury. First, we analyzed the HSC clonogenic via a cobblestone area-forming cell (CAFC) assay. The 35-day CAFC in the vehicle-treated mice was lower than the control mice. However, the injury. First, analyzed the HSC clonogenic function a cobblestone area-forming cell (CAFC) assay. Theinwe 35-day CAFC in the vehicle-treated micerelieved wasvia lower than the control mice. However, decrease the HSC clonogenic function was not by SB203580 treatment (Figure 3A). the To assay. The 35-day CAFC in the vehicle-treated mice was lower than the control mice. However, decrease in the HSC clonogenic function was notcolony relieved by SB203580 3A). To further validate this result, we used a single-cell assay. Similarly,treatment the results(Figure demonstrated thefurther decrease in thethis HSC clonogenic function was not relieved by SB203580 treatment (Figure 3A). validate result, used a single-cell assay. Similarly, the results demonstrated that SB203580 treatment didwe not rescue the HSC colony clonogenic function suppression induced by TBI, To that further validate this result, we used a single-cell colony assay. Similarly, the results demonstrated SB203580 treatment did not rescue the (Figure HSC clonogenic function suppression by does TBI, which was consistent with the CAFC assay 3B). These findings suggest thatinduced SB203580 that SB203580 treatment did not rescue the HSC clonogenic function suppression induced by TBI, which was consistent with the CAFC assay (Figure 3B). These findings suggest that SB203580 does not attenuate TBI-induced long-term HSC suppression. not attenuate TBI-induced long-term HSC (Figure suppression. which was consistent with the CAFC assay 3B). These findings suggest that SB203580 does not attenuate TBI-induced long-term HSC suppression.
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Figure 3. 3. Effects of of SB203580on onTBI-induced TBI-inducedlong-term long-term BMinjury. injury. Mice were treated with vehicle Figure treated with vehicle or FigureEffects 3. Effects SB203580 of SB203580 on TBI-induced long-termBM BM injury. Mice Mice were were treated with vehicle or SB203580 70 days after exposure to 6 Gy TBI, as described in the experimental section. BMNCs SB203580 70 days after exposure to 6 Gy TBI, as described in the experimental section. BMNCs were or SB203580 70 days after exposure to 6 Gy TBI, as described in the experimental section. BMNCs were collected from the days mice after 80 days 6 Gy TBI. (A) The clonogenic function of HSCs was collected the mice Gyafter TBI. TheTBI. clonogenic of HSCs was were from collected from 80 the mice 80 6 days after(A) 6 Gy (A) The function clonogenic function ofmeasured HSCs wasvia a measured via(B) a The CAFC assay; capacity ofHSCs HSCsinusing inBM BM was analyzed using CAFC assay; clonogenic capacity of HSCs in capacity BM was of analyzed awas single-cell colony assay. measured via a CAFC assay;(B) (B)The The clonogenic clonogenic analyzed using a a single-cell colony assay. single-cell colony assay.
2.4. SB203580 Inhibits TBI-Induced Chronic Oxidative Stress 2.4. 2.4. SB203580 Inhibits TBI-Induced Stress SB203580 Inhibits TBI-InducedChronic ChronicOxidative Oxidative Stress Increasing evidence demonstrates that total-body exposure to radiation in mice induced long-term Increasing evidencedemonstrates demonstrates that that total-body total-body exposure in in mice induced Increasing evidence exposuretotoradiation radiation mice induced BM suppression via the induction of chronic oxidative stress and senescence in HSCs [16,17]. Moreover, long-term suppression viathe theinduction inductionof of chronic chronic oxidative and senescence in HSCs [16,17]. long-term BMBM suppression via oxidativestress stress and senescence in HSCs [16,17]. exposure to a sub-lethal dose of TBI selectively induced high levels of intracellular ROS in HSCs [16,18]. Moreover, exposure to a sub-lethal dose of TBI selectively induced high levels of intracellular ROS in in Moreover, exposure to a sub-lethal dose of TBI selectively induced high levels of intracellular ROS The HSCs production ofThe ROSproduction was increased in the HSCs ratherinthan the HPCs from the vehicle-treated mice, [16,18]. of ROS was increased the HSCs rather than the HPCs from the HSCs [16,18]. The production of ROS was increased in the HSCs rather than the HPCs from the evenvehicle-treated 80 days after mice, 6 Gy TBI, compared with the control mice (Figure 4), which suggests that persistent even 80 days after 6 Gy TBI, compared with the control mice (Figure 4), which vehicle-treated mice, even 80 days after 6 Gy TBI, compared with the control mice (Figure 4), which oxidative stress in oxidative HSCs 80 days TBI. suggests thatexisted persistent stressafter existed in HSCs 80 days after TBI. suggests that persistent oxidative stress existed in HSCs 80 days after TBI.
Figure 4. Effects of SB203580 on the TBI-induced increase in ROS in HPCs and HSCs. Mice were Figure 4. Effects of SB203580 on the TBI-induced increase in ROS in HPCs and HSCs. Mice were treated treated with vehicle or SB203580 70 days after exposure to 6 Gy TBI, as described in the with vehicle or SB203580 70 days exposure to 6increase Gy TBI, in as described in theand experimental section. Figure 4. Effects of SB203580 onafter the TBI-induced ROS in HPCs HSCs. Mice were experimental section. BMNCs were collected from the mice 80 days after 6 Gy TBI, and the levels of BMNCs were collected from the mice 80 days after 6 Gy TBI, and the levels of intracellular ROS in treated with vehicle or HSCs SB203580 70 days exposure to are 6 presented Gy TBI, as as the described in the intracellular ROS in the and HPCs were after measured. The data means ± SD. the HSCs and HPCs were measured. The data are presented as the means ˘ SD. n = 12 mice/group; experimental section.aBMNCs were collected from 80 days after 6 Gy TBI, and the levels of b p < 0.05 p < 0.05 vs. control; vs.the TBImice + vehicle. n = 12 mice/group; a p < 0.05 vs. control; b p < 0.05 vs. TBI + vehicle. intracellular ROS in the HSCs and HPCs were measured. The data are presented as the means ± SD. a pp38 b p < 0.05 < 0.05 vs. control; vs. TBI vehicle. n = SB203580 12 mice/group; 2.5. Inhibits Expression Augment in HSCs but+ Not Senescence in HSCs
2.5. SB203580 Inhibits p38 Expression Augment in HSCs but Not Senescence in HSCs Research has demonstrated that p38 is crucial for the maintenance of HSC quiescence because it 2.5. SB203580 Inhibits p38 Expression Augment in HSCs but Not Senescence in HSCs may be activated by ROS [19]. that Furthermore, p38 isfor involved in the mediation of quiescence radiation-induced Research has demonstrated p38 is crucial the maintenance of HSC because it senescence, which is an element attributable to long-term BM injury Previous studiesbecause have it mayHSC be activated ROS [19]. Furthermore, p38 is involved in the mediation of radiation-induced Research hasby demonstrated that p38 is crucial for the maintenance of[9]. HSC quiescence demonstrated the vital role of p38 in ROS regulation[16]. The increased levels of ROS led to the HSC senescence, which an element attributable to long-term injury [9]. Previous studies may be activated by ROSis[19]. Furthermore, p38 is involved in theBM mediation of radiation-induced induction of HSC senescence, as they expressed a higher level of p16 expression (Figures 5A,B). have demonstrated the vital of p38 in ROS regulation [16].BM The increased levels ofstudies ROS led to HSC senescence, which is an role element attributable to long-term injury [9]. Previous have Treatment of with SB203580 inhibited theexpressed TBI-induced induction of ROSexpression production(Figure in HSCs the induction HSC senescence, as they a higher level of p16 5A,B). demonstrated the vital role of p38 in ROS regulation[16]. The increased levels of ROS led to the (Figure 4); however, SB203580 did not reduce the HSC expression of p16 expression. To determine Treatment SB203580 inhibited the TBI-induced of ROS production in HSCs (Figure 4); induction with of HSC senescence, as they expressed a induction higher level of p16 expression (Figures 5A,B). Treatment with SB203580 inhibited the TBI-induced induction of ROS production in HSCs (Figure 4); however, SB203580 did not reduce the HSC expression of p16 expression. To determine
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however, SB203580 notHSC reduce the HSC expression of p16 expression. To determine whether SB203580did affects function via the ROS-p38-p16 pathway, we measured the levelwhether of p38 SB203580 affects HSC function via the ROS-p38-p16 pathway, we measured the ofand p38 phosphorylation (p-p38) in HSCs. TBI induced a substantial increase in p38 activation inlevel HSCs, phosphorylation (p-p38) in HSCs. TBI induced a substantial in TBI, p38 compared activation with in HSCs, SB203580 treatment reduced the p-p38 expression level 80 daysincrease after 6 Gy the andvehicle SB203580 treatment reduced p-p38 expression level days after 6 Gy TBI-induced TBI, compared with the treatment (Figure 5A).the These findings suggest that80SB203580 affects long-term BM treatment suppression probably inhibiting TBI-induced chronic oxidative and increases in vehicle (Figure 5A).by These findings suggest that SB203580 affectsstress TBI-induced long-term expression, whichby is the ROS-p38TBI-induced pathway; however, not ameliorate senescence BMp-p38 suppression probably inhibiting chronicSB203580 oxidativedid stress and increases in p-p38 in HSCs.which is the ROS-p38 pathway; however, SB203580 did not ameliorate senescence in HSCs. expression,
Figure 5. Effects of SB203580 onon TBI-induced HSCs. Mice Micewere weretreated treated Figure 5. Effects of SB203580 TBI-inducedincrease increasein inROS ROSin in HPCs HPCs and HSCs. withwith vehicle or SB203580 70 days after after exposure to 6 Gy described in the experimental section. vehicle or SB203580 70 days exposure to TBI, 6 GyasTBI, as described in the experimental section. BMNCs werefrom collected from80 thedays miceafter 80 days Gy TBI, and HSCs were isolated from BMNCs were collected the mice 6 Gyafter TBI,6and HSCs were isolated from BMNCs BMNCs by cell sorting. (A,B) Analysis of theofexpression p-p38 and p16 HSCs. Left: by cell sorting. (A,B) Analysis of the expression p-p38 and of p16 in HSCs. Left:inrepresentative representative photomicrographs p-p38/p16 DAPI immunostaining nuclear staining in immunostaining photomicrographs of p-p38/p16 andofDAPI nuclearand staining isolated HSCs in are isolated HSCs shown; Right: the percentages ofHSCs p-p38/p16 positive HSCs presented shown; Right: theare percentages of p-p38/p16 positive are presented as theare means ˘ SD as (n the = 3). means ± SD 10ˆ; (n = 3). 10×; mRNA (C) Theexpression levels of p16 expression in the HSCs were Magnification: (C)Magnification: The levels of p16 in mRNA the HSCs were analyzed via RT-PCR. analyzed via RT-PCR. The data are presented as the means ± SEM of the fold changes compared The data are presented as the means ˘ SEM of the fold changes compared with the control (n = 3). b ap< with (n b= p3).
