Curcumin Promotes Antioxidative Stress Of Mouse Bone Marrow Stem Cells Through Mtor Signal Pathway

BMSCs(Bone Marrow Stem Cells), an ideal seed cells of tissueengineering, have powerful reproductive and multi-directionaldifferentiation potential, as well as its advantage of autograft ofectogeneses gene transfection, and is not related to the issues of ethics andso on, therefore, BMSCs have became more and more attention by people.And now BMSCs are used as cell therapy in some clinical diseases that areineffectively treated by traditional approaches, such as dysostoses, acuteand chronic graft versus host reaction, inflammatory bowel disease,ischemic heart disease, unhealing ulcer, ischemic stroke, parkinson’sdisease and so on. Although the cell therapy provid a new therapeutic tooland takes new hope for many clinical disease, the further study shows thatthe survival ratio was very low and almost99%of BMSC becomeapoptosis after transplanted for24h[1]. Hence the low survival ratio becomea key barrier for cell transplantation.The ischemia and hypoxia as will as inflammatory reflects ofmicroenvironment in the damage local (ROS major come from neutrophiland macrophage) can cause the large amount release of ROS(reactiveoxygen species). Finaly cell apoptosis, necrosis, and tissue damage areinduced. The reactive oxygen species including: superoxide anion,hydroxyl radical, hydrogen peroxide (H2O2) and so on, they can lead to cell death through the activation of multiple signal transduction pathways.Therefore, improving the ability of antioxidation stress damage of BMSCsis the key problem to solve the low survival of BMSCs transplant.Among the numerous pathways of oxidative stress,(mammalian targetof the rapamycin) mTOR pathway plays an important role in cell growthand preventing cell death, as well as the control of cell apoptosis[2]. Manystudies show that the micromolecule inhibitors of mTOR signal canpromote apoptosis through reducing expression of Mcl-1and increasing theexpression of phosphorylated Bim. Bim can directly stimulate theactivation of Bax and Bak, and indirectly combine to the anti-apoptosisprotein such as Bcl-2,Bcl-XLand Mcl-1, meanwhile, can also inhibit themTOR signal pathway, resulting in cell apoptosis[3].Curcumin is know as a base of the diet spice, and is a polyphenolnatural product isolated from the rhizome of the plant Curcuma longa.Extensive studies demonstrated curcumin has antioxidation,anti-inflammatory, anticancer, anti-amyloid properties and so on. Recentyears, curcumin has been shown to therapy various chronic illnesses, suchas Alzheimer’s disease(AD), Parkinson’s disease, multiple sclerosis,epilepsy, cerebral injury, diabetes, AIDS and so on. The mechanism of thecurcumin are recognized:①antioxidation: oxidative damage is one of thedestructive stimulus as regarded to BMSCs transplant therapy. Curcumin asa natural antioxidant, its phenols hydroxyl which can directly capture orclear free radicals, this ability is even stronger than vitamin.②anti-inflammatory reaction: inflammatory reaction is also one of the majorreason of damage in stem cell transplantation. In vitro experiments showthat curcumin may clear free radical, and protect lipids and proteins awayfrom oxidative damage. But we haven’t seen the report that whether thecurcumin can play the antioxidation through mTOR pathway, when the BMSCs are exposed to oxidative stress.To explore the change of the morphology of BMSCs and the mTORsignal pathway under the oxidative stress injury, BMSCs were treated withdifferent concentration of H2O2. the activity of BMSCs was detected byMTT. The expression of mTOR signal pathway proteins and the apoptosisproteins was analyzed by Western blot. To determine whether curcuminameliorates H2O2–induced the oxidative stress injury on BMSCs, weexamine the expression of mTOR signal pathway proteins and theapoptosis proteins by western blot analysis. Our results may provide novelinsights into the mechanisms by which curcumin inhibits the H2O2-inducedBMSCs oxidative stressPart one: the effect of mTOR signal pathway and apoptosisassociated protein in oxidative stress of BMSCsMethods1. Isolation, cultivation and identification of BMSCs2. Establishment and identification of oxidative stress model ofBMSCs. Experimental groups: the control group(BMSCsroutine culture) and oxidative stress injury groups (BMSCsroutine culture+treated with H2O2of100、200、300、400、500、800、1000μmol/L, respectively. n=5).3. Oxidative stress stimulation damage on BMSCsThe morphology change of BMSCs treated with H2O2for24h wasobserved by inverted phase contrast microscope.Proliferation of BMSCs treated with H2O2for24h,48h,72h wasdetected by MTT assay.Apoptosis morphological changes of BMSCs treated with H2O2for24h were observed by fluorescence microscope. 4. Western blot was used to detect the protein andphosphorylated protein expression of mTOR, p70s6k, s6BMSCs were treated with H2O2.5. The expression of Bax, Bcl-2protein were detected bywestern blot, after BMSCs were treated with H2O2.6. All the data were analyzed with the SPSS17.0, andcomparison between groups were tested by One-WayANOVA analysis and LSD test.Result1. Altered pathologic morphology of BMSCs occurred coincident withH2O2concentration, after the treatment with H2O2of100～1000μmol/L for24h.2. The result of MTT assay shows that the death number of BMSCstended to be increased as a dose dependence and time dependencemanner.3. The result of hochest33342staining shows: the apoptosis of BMSCswere increased, compare to the control group, the number ofapoptosis cell increased significantly after H2O2treatment withconcentration of500～1000μmol/L.4. The protein expression of mTOR, P70S6K, S6in BMSCs tended tobe increased after treatment with H2O2at the concentration of100～300μmol/L, while the expression of their phosphorylateprotein and anti-apoptosis protein Bcl-2were increasedsignificantly(P﹤0.01), Meanwhile, the expression of apoptosisprotein Bax was not obviously changed(P﹥0.05).5. A dose-dependent decrease was found in p-mTOR, p-P70S6K,p-S6,Bcl-2protein after treatment of BMSCs with H2O2(400～ 1000μmol/L)（P﹤0.05，P﹤0.01）, while the expression of mTOR,P70S6K, S6protein was not visibly altered, whereas the expressionof apoptosis protein Bax was significantly increased（P﹤0.01）.conclusionOxidative stress to some extent causes reduced the survival ratioand increased the cell apoptosis in BMSCs. The underlying mechanismsmay be partly due to the inhibition of mTOR and its downstream signalpathway, and decreased expression of anti-apoptosis protein Bcl-2withenhanced expression of apoptosis protein Bax.Part two: the effect and mechanism of curcumin on BMSCs inoxidative stressmethod1. Grouping:1) control group(conventional culture of BMSCs,with no H2O2),2) oxidative stress damage group(control group+treated with H2O2of500μmol/L),3) curcumin group(treatedwith curcumin of3.125,6.25,12.5,25,50μmol/L,respectively),4)rapamycin and curcumin group (oxidative stress damage group+treated with rapamycin of50ng/mL and curcumin of12.5μmol/L).2. The antioxidation of different concentration of curcumin onBMSCs was detected by MTT.3. The protein expression of Bax, Bcl-2as well as thephosphorylate protein expression of mTOR, p70s6k, s6inBMSCs in all groups were detected by western blot.4. All the data were analyzed with the SPSS17.0, and groupcomparisons were tested by One-Way ANOVA analysis andLSD test. Result1. The result of the MTT assay shows that the curcumin of12.5μmol/L play strongest antioxidative stress effect.2. Compare to the oxidative stress damage group, the proteinexpression of p-mTOR, p-P70S6K, p-S6were increased at theconcentration of curcumin at12.5μmol/L（P﹤0.01）; but theseeffect can be block by rapamycin(a mTOR signal pathwayinhibitor) significantly（P﹤0.01）.3. Compare to the oxidative stress damage group, the expression ofBcl-2protein was increased at the concentration of curcumin at12.5μmol/L（P﹤0.01）, and the expression of Bax decreased（P﹤0.01）; but these effect can be blocked by rapamycin(a mTORsignal pathway inhibitor) significantly.ConclusionCertain concentration of curcumin can play a role of antioxidativestress, the underlying mechanisms may involve the activation of mTORsignal pathway, the up-regulation of antiapoptosis protein Bcl-2, and theinhibition of proapoptosis protein Bax.