COX-2 Inhibition Deteriorates Radiation-induced Damage In Mice Colonic Mesenchymal Stem Cells

Background and purposeIonizing radiation can cause damage to organs or tissues.Many organs or tissues are sensitive to ionizing radiation,including immune and hematopoietic organs,gastrointestinal tract,lung,brain,and so on.Among the various body systems,gastrointestinal tissues are,subsequent to immune and hematopoietic systems,one of the most sensitive parts to radiation toxicity.And radiation-induced enteropathy has become a hot topic in radiobiological field.After being exposed to ionizing radiation,a serial of pathological changes may happen in the gastrointestinal tract,mainly exhibit as increasing of epithelial cell apoptosis,decreasing of crypt survival,followed by damage to mucosal integration and dysfunction of gastrointestinal tract.Statistically,it is reported that the morbidity of enteropathy provoked by irradiation has exceeded that of inflammatory bowel diseases（IBD）,very common diseases in digestive system.Wide attention has been drawn to radiation-induced enteropathy,and imperative efforts should be done to conquer such a severe clinical problem.It is widely reported that cyclooxygenase-2（COX-2）contributes to protecting intestinal tissues from radiation-induced injury.Recent researches find that a group of mesenchymal stem cells（MSCs）,though in small populations,existing around the intestinal crypts contributes substantially to ameliorating radiation-induced enteropathy.These cells are featured as constitutive expression of cyclooxygenase-2（COX-2）,through which the radioprotection role of the remaining mesenchymal stem cells to the intestinal tissues comes into reality.Thus,MSCs has great potential in protecting intestine from radiation-induced damage.As is known to us all,cyclooxygenases inhibitors（COXIs）,including aspirin and celecoxib,are in wide application in clinical practice.The application of these drugs is very common in disease prevention and treatment,such as inflammatory diseases,rheumatoid arthritis,and inhibition of platelet aggregation.However,these drugs share common pharmacological effect,that is inhibiting biological activity of COX-2 enzyme.Given the important role of intestine-specific MSCs in protecting intestinal tissues from radiation injury via COX-2 signaling,we wonder whether those widely application agents（i.e.aspirin and celecoxib）,with efficacy in inhibiting COX-2 enzyme bioactivity,exert side effects to deteriorate radiation-induced injury to those intestine-derived MSCs,and whether these drugs will have influence on intestinal tract after radiation exposure.In this study,we focus on MSCs in the intestinal tract,aim to establish a method for isolation and culture of intestinal MSCs in vitro,then investigate the role of COX-2 inhibitors in radiation-induced damage to intestinal mesenchymal stem cells,and explore the effect of COX-2 inhibitors,i.e.aspirin and celecoxib,on organ or tissue damage provoked by ionizing radiation in mice,so as to establish the basic experimental conditions for futher investigating the role of these drugs on radiation-induced in vivo.This work will contribute to research and development of radioprotectors with novel ideas and fundamental basis.Meanwhile,our efforts will help to provide important evidence for clinical rational drug use.Methods1.Isolation and culture of MSCs from mice colonic tissuesA two-step method was established to extract MSCs from mice colons.Cells were maintained in low-glucose DMEM.2.Cell characterizationCell growth status and morphological feature were observed under microscope.Flow cytometry was used to detect phenotypic profile of isolated colonic mesenchymal stem cells;COX-2 expression was detected by immunocytochemistry,western blot and flow cytometry analysis.CCK-8 analysis was used for analysis of cell proliferation capacity.Cell stemness was detected by differentiation induction in vitro and real-time PCR analysis for relevant gene expression.3.Screening of drug concentrationColonic MSCs in subculture were stimulated with various concentrations of aspirin or celecoxib for 24 h,then cell viability in each group was detected using CCK-8 analysis,and the proper concentration of the two drugs were chosen for the following in vitro assay.4.Effect of aspirin or celecoxib on COX-2 expression in MSCs from mice colonsCells were stimulated with selected concentration of aspirin or celecoxib for 24 h and 48 h,then COX-2 expression in each group of MSCs was detected using western blot analysis.5.In vitro radiation modela.Cytotoxic effect of different doses of ⁶⁰Co-γ rays on MSCs from mice colonsCells were subjected to various doses of ⁶⁰Co-γ ray irradiation,24 h after radiation exposure,cell proliferation in each group was detected using CCK-8 analysis,and the proper dose of radiation exposure was determined for the following in vitro assay.b.Cytotoxic effect of ⁶⁰Co-γ rays on cMSCs at different time after radiation exposureColonic MSCs in subculture were subjected to 15 Gy of ⁶⁰Co-γ ray irradiation,cell proliferation at different time after irradiation was detected using CCK-8 analysis,and the proper time for endpoint analysis was determined for the following in vitro assay.6.Effect of aspirin and celecoxib on radiation-induced damage to MSCs from mice colonsColonic MSCs from mice were pre-treated with asipirin（75 μmol/L）and celecoxib（20 μmol/L）for 24 h,then cells were subjected to a single dose of 15 Gy of ⁶⁰Co-γ ray irradiation,and 24 h later,cell proliferation were detected using CCK-8 analysis.7.Effect of COX-2 inhibitors on C57BL/6 mice after radiation exposureMice were administered with aspirin（15 mg/kg/d）,celecoxib（60 mg/kg/d）,or equivalent vehicle by gavage for a consecutive of 2 weeks,then mice in radiation group were subfected to a single dose of total body irradiation（TBI）using gamma 60 Co irradiator.After radiation exposure,mice were sacrificed at the indicated time.Histological changes and tissue injury were then analyzed and compared between different groups.1)Intestinal tractMice were sacrificed at the indicated time,and both small and large intestinal tissues were taken out promptly.The proximal jejunal and colonic segments were fixed in 4% PFA for histological analysis by HE and TUNEL staining.For analysis of intestinal crypt survival,120 mg/kg BrdU agent in saline was given to each animal intraperitoneally 2 hours ahead of being sacrificed.2)Thymus and spleensAt the indicated endpoint after being irradiated,mice were sacrificed,following body weight recording,and thymuses and spleens were taken out promptly.The weight of these organs was then recorded,and relevant organ weight index were obtained.3)Bone marrowMice were sacrificed at the indicated time,and femurs were taken out promptly and fixed in 4% paraformaldehyde（PFA）.Both HE and TUNEL staining were used to detect histological changes in mice bone marrows of each group.Results1.Isolation,culture and characterization of cMSCs in mice1)Basic features of mesenchymal cells isolated from mice colonsDuring culture,plastic adherent cells were easy to be seen under microscope.Morphologically,these colonic mesenchymal cells were fibroblast-like spindle shape.And there were no observed changes in cell morphology during subculture of these cells.2)Phenotypic expression of CD29 and CD44 in mesenchymal cells extracted from mice colonsCD29 and CD44 surface markers were found in cultured passage 3 mesenchymal cells from mice colons as indicated by flow cytometry analysis.Of the two phenotypic markers,CD29 positive cells exhibited a little more than CD44,with percentages of（97.48 ± 1.18）% and（91.18 ± 1.97）% respectively.3)Proliferative capacity of mesenchymal cells extracted from mice colonsCCK-8 analysis demonstrated that cell proliferation rate increased with the prolonging of culture period.4)Differentiation capacity of mesenchymal cells extracted from mice colonsAlizarin Red S staining showed osteocytes formation and Oil Red O staining showed adipocytes formation in mice colon-derived mesenchymal cells after being cultured in differentiation induction medium for a period of time.5)Gene expression concerning cell stemness in mesenchymal cells extracted from mice colonsBmi 1 and Lrig 1 were likely expressed in mesenchymal cells from mice colons as indicated by real time-PCR analysis of genes relevant to cell stemness.6)COX-2 expression in mesenchymal cells extracted from mice colonsInherent expression of COX-2 intracelllularly in cultured mesenchymal cells from mice colonic tissues.Western blot and flow cytometry analysis also detected COX-2 in these colon-derived mesenchymal cells.2.Effect of aspirin and celecoxib on normal cMSCs in vitro1)Effect of different concentrations of aspirin and celecoxib on cell viability in colonic mesenchymal stem cellsCCK-8 analysis showed that aspirin did not present obvious cytotoxic effect to mice colon-derived mesenchymal stem cells,even at the highest concentration of 100 μM.No significant cytotoxic effect was observed in relatively low concentrations of celecoxib.When compared with control group,cell viability in group of 20 μM celecoxib showed no significant change [（94.58 ± 7.04）% vs（100.00 ± 6.01）%,P>0.05].2)Effect of selected concentration of aspirin and celecoxib on COX-2 expression in cMSCsWestern blot analysis showed no significant changes of COX-2 expression between each groups.3.Effect of ⁶⁰Co-γ ray irradiation on cMSCs in vitro1)Proliferation inhibition effect of different radiation doses on cMSCsCCK-8 analysis showed that ⁶⁰Co-γ ray irradiation did play a certain role in inhibiting cell proliferation of in vitro cultured mammalian colon-derived MSCs.Significant deduction of cell proliferation rate was observed,when compared to control group,in colonic mesenchymal stem cells irradiated by 15 Gy ⁶⁰Co-γ ray [（86.97 ± 6.61）% vs（100.00 ± 4.53）%,P<0.01].2)Cell proliferation rate of cMSCs at different time after radiation exposureCCK-8 analysis showed that there was no significant deduction of cell proliferation rate,compared to control group,in in vitro cultured colonic mesenchymal stem cells 6 h after radiation sitimulation [（95.62 ± 4.57）% vs（100.00 ± 1.53）%,P>0.05].However,an obvious decrease of cell proliferation rate was observed 24 h after radiation exposure [（89.90 ± 2.16）% vs（100.00 ± 1.53）%,P<0.01].Whereas,this proliferation inhibiting effect of ⁶⁰Co-γ ray on in vitro cultured mice colonic mesenchymal stem cells did not increasing more even the post-irradiation time reached to 72 h（P>0.05）.4.Effect of COX-2 inhibitors on radiation damage to colonic mesenchymal stem cellsCCK-8 analysis showed significant deduction of cell proliferation rate in cells of radiation group,as compared to control group [（90.22 ± 1.70）% vs（100.00 ± 5.37）%,P=0.012].Aspirin pretreatment did not aggravate radiation-induced MSCs injury [（86.67 ± 4.94）% vs（90.22 ± 1.70）%,P=0.321].However,an obvious decrease was observed in radiation + celecoxib group [（80.86 ± 0.96）% vs（90.22 ± 1.70）%,P=0.025].5.Effect of COX-2 inhibitors on the intestinal tract exposed to ionizing radiation in C57BL/6 miceHE staining showed that there were no obvious changes on mice small intestinal villus and colonic mucosa in groups stimulated with aspirin,celecoxib and irradiation,as compared to those of control group.Besides,no significant changes in crypt survival of the whole intestine were observed in any group of mice as indicated by immunohistochemical staining.The above results indicated that these two cyclooxygenase-2 inhibitors in the given doses have no significant toxic effect on the intestinal tract of C57BL/6 mice.6.Effect of COX-2 inhibitors on thymus and spleen exposed to ionizing radiation in C57BL/6 miceWhen compared to control group,mice in 4 Gy irradiation group showed significant decrease in weight index of thymus 3.5 d post-irradiation [（34.11±5.74）% vs（100.00±16.37）%,P<0.01],with similar result for spleen [（35.30±4.71）% vs（100.00±10.30）%,P<0.01].The weight index of thymus decreased much more in high dose irradiation group than that of low dose irradiation group at the time point of 5.5 d [（13.19±1.96）% vs（40.40±13.46）%],with no obvious changes in the weight index of spleens [（30.28±1.13）% vs（31.20±1.17）%].When it came to high dose irradiation,both thymus and spleens exhibited improved weight index 7 d post irradiation when compared to that of 5.5 d,with [（34.03±8.40）% vs（13.19±1.95）%] for thymus,and [（37.32±2.10）% vs（30.28±1.13）%] for spleen.When compared to control group,no significant decline in weight index of the two immune organs was observed in mice pretreated with either aspirin or celecoxib.7.Effect of COX-2 inhibitors on the bone marrow exposed to ionizing radiation in C57BL/6 miceHE staining showed that ⁶⁰Co-γ ray irradiation caused significant damage to mice hematopoietic system,mainly presented as decreased cells in bone marrow as well as increased vacuolation formation.What’s more,dramatic apoptotic cells were observed in mice bone marrow after total body irradiation as indicated by TUNEL staining.When compared to control group,no obvious pathological changes of bone marrow were observed in mice pretreated with either aspirin or celecoxib.Conclusions1)We successfully established the method to culture intestinal MSCs in vitro;2)These MSCs from mice colonic tissues showed constitutively expression of cyclooxygenase-2 intracellularly;3)⁶⁰Co-γ ray irradiation did have certain cytotoxic effect on intestinal-derived mesenchymal stem cells from C57BL/6 mice;4)Celecoxib,a selective COX-2 inhibitor,could aggravate intestinal-derived MSCs injury provoked by ionizing radiation;5)Aspirin（15 mg/kg/d）and celecoxib（60 mg/kg/d）have no significant side effect on thymus,spleen,bone marrow and gut in normal C57BL/6 mice;6)Both 4 Gy and 8 Gy ⁶⁰Co-γ ray total body irradiation can lead to severe pathological changes in thymus,spleen,and bone marrow in C57BL/6 mice,but not intestinal tract;7)Aspirin and celecoxib pretreatment did not deteriorate organ or tissue injury after radiation in mice.