The Impact Of Small-molecule Compounds On The Senescence Of Bone Marrow Mesenchymal Stem Cells During The Process Of Bone Aging

In the aging process, bone is aging gradually with the increase of age. Aging boneleads to occur of osteoporosis, and results in catagma, hairline fracture and other diseases.China is the world’s most populous country in the elderly, and about90million peoplesuffer from different degrees of osteoporosis. So it is of great significant in the preventionand the treatment of osteoporosis. In the process of bone aging, the speed of boneresorption by osteoclasts outstrips the speed of bone regeneration by osteoblasts, whichmeans the balance of bone metabolism is broken and results in osteoporosis. Bone marrowstromal cells(BMSCs), as the precursor cells of osteoblasts, is the key factor in boneregeneration. Proliferation potential and differentiation potential of BMSC is decreasingwith the aging process, that is the main reason for the development of osteoporosis. At present, drug treatment of osteoporosis is mainly focused on regulation ofosteoblasts/osteoclasts. Disadvantages of such treatment are lack of therapeutic target,severe side effects and financially expensive in the long run. Therefore, the close linkbetween BMSC and osteoporosis means that regulation of BMSCs has a broad prospect inthe field.Small-molecule compounds, hotspots in recent years, are wildly researched in theregulation of cell. China has a rich variety of natural compounds, which means a greatpreponderance in the field of small-molecule compounds based stem cell treatment. Onthe basis of this, our study is focus on the screening of small-molecule compounds whichhas a reversing effect on the aging process of BMSC in the process of bone aging.Through the exploration of molecular mechanism and pharmacological activity, we wantto offer the necessary research foundation of the drug treatment in osteoporosis. Based onthe library of new-found small-molecule compounds, we selected several kinds ofcompounds which have an obvious impact on the regulation of BMSC’s fate. Then wedetermined more about its molecular mechanism and effects in vivo, and successfullydiscovered the ideal compounds.1.Scheme1)In the first part of the study, we made a screening of the about14’000small-molecule compounds from the24compounds library. First, we minimized our rangeof the screening by the activities of the small-molecule compounds provided by theNational Chemical Library Centre. Then we analysed recent studies and related targets ofthe compounds to make our selection. A total of27kinds of small-molecule compoundswere chosen for the next step of our studies, which are not researched in the related fieldand have the potential to interfere the stem cells.2)Next, we gain aging-BMSCs from aged male SD rats for our compounds treatment.The chosen small-molecule compounds were added to the aging-BMSCs at gradientconcentration of1000μM、100μM、10μM、1μM. RT-PCR test was performed to detect thechange of aging related targets of BMSC interfered by the compounds in mRNA level. Here we used β–gal、p53、p21、p16、Nanog、Oct-4、TERT as our targets of screeningwhich are closely linked with the aging process of BMSCs. By the results of this step, wedetermined a total of11kinds of small-molecule compounds which have a significanteffect on the aging-BMSCs and their ideal concentration. Then a crosswise comparison ofthe11kinds of compounds was made using cells from the same batch, and the effect wasevaluated. Finally,5compounds （apocynin、bax channel blocker、iso-Olomoucine、（±）bay k8644、H-9） was coming through for the next step of study in advantage of theiroutstanding effect.3)In the third step, we treated aging-BMSCs with chosen5compounds respectivelyat their ideal concentration, and a variety of tests was performed to determine the changeof cell behavior. MTT test was made to determine the potential of proliferation of BMSC;SA-β–gal staining was made to detecting aging level of the cells; RT-PCR and westernblot test were made to examine changes in aging and differentiation at mRNA level aswell as protein level. ALP staining, quantitative analysis of ALP activity and alizarin redstaining were made to examine changes in osteogenic differentiation; flow cytomery testwas performed to examine cell cycle and cell apoptosis. Results of these studies show thatall5compounds have a significant effect in reversing aging process of aging-BMSCs.4)At last, we made an in-depth analysis of apocynin. On the one hand, we explore themolecular mechanism of apocynin in vitro. NADPH oxidase quantitative analysis,intracellular ROS level detection were performed to discover ways how apocynin changethe aging process of BMSC. On the other hand, SAMP6mice model was selected for theevaluation of apocynin in vivo. And from this part, we determined that apocyninsignificantly improved osteoporosis in SAMP6mice, as well as the bone aging process.Micro-CT scanning was made to examine changes in BMD and total bone value;TRAP-ALP staining was performed to examine changes in the balance ofosteoblasts-osteoclasts. At the same time, BMSCs of SAMP6was gained for examinationin vitro.2.Results Part I Selection of small-molecule compounds1)Through analysis of libraries provided by National Chemical Library Centre, weselected8libraries for our screening in which activities of compounds were listed. Andour screening follows particular standard.2)According to the activities provided by the list, a total of27kinds ofsmall-molecule compounds was selected for the next studies.Part II Initial screening of the selected small-moleculecompounds1)Aging-BMSCs was obtained from22months old male SD rats. Cells weredetermined in aging characteristic, to make sure that cell model for screening fit ourrequirement.2)All27compounds were diluted into different concentrations (1000μM、100μM、10μM、1μM), and BMSCs were treated by them respectively. Cells treated by compoundswere then tested in RT-PCR. β–gal、p53、p21、p16、Nanog、Oct-4、TERT were testedto determine the effect level in aging process. According to this result,11kinds ofcompounds was chosen for the next study and their ideal concentration were determined.3)Then a crosswise comparison was made in the same batch of cells. RT-PCR testwas performed to determine the effect of compounds. Apocynin、bax channel blocker、iso-Olomoucine、（±）bay k8644、H-9were chosen for the following study for theirsignificant effect on aging process.Part III Effect of small-molecule compounds on aging-BMSCs1)In examination of aging-related targets of BMSCs, apocynin、bax channel blocker、iso-Olomoucine、（±）bay k8644、H-9all have a significant effect on the targets at differentdegree.2)Osteogenic differentiation was induced in aging-BMSCs, and the chosensmall-molecule compounds was added into the process of differentiation. In theexamination of differentiation related targets, we found that apocynin、bax channel blocker、iso-Olomoucine、（±）bay k8644all have a improvement in different stages of theosteogenic differentiation.Part IV Molecular mechanism of apocynin and its effect in vivo1)We first explored the molecular mechanism of apocynin in BMSCs aging process.Results showed that NADPH oxidase activity was inhibited by apocynin, and intracellularROS was reduced as well. Then we examined the relation between the change ofintracellular ROS affected by apocynin and expression of p53. Results showed thatexpression of p53was positive related to the intracellular ROS level.2)SAMP6mice model was chosen for experiments in vivo. Mice at3months wereinjected with apocynin in intraperitoneal and the injection was done3times a week.3months after injection, long bones was obtained for the analysis. Result of micro-CTshowed that bone value was improved by apocynin. TRAP-ALP result showed thatosteoblasts were enhanced by apocynin.3)BMSCs from SAMP6mice were gained for experiments in vitro. SA-β–gal resultsshowed that degree of aging was inhibited by apocynin in late passages. In mRNA level,RT-PCR results showed that p53was inhibited and expression of oct-4was enhanced.After osteogenic differentiation, apocynin group maintained a higher activity of ALP thannegative control group.3. ConclusionApocynin、bax channel blocker、iso-Olomoucine、（±）bay k8644were determinedto have a significant reversing effect on the aging process of BMSCs. All4compoundsimproved aging-related gene expression at cellular level, and proliferation andosteogenesis showed improvement as well. We can draw a conclusion that it is of greatsignificance to continue the study of these compounds in field of stem cell treatment ofosteoporosis.We also determined that apocynin reduced intracellular ROS level by inhibitingactivity of NOX, which directly resulted in expression of p53. Then a series of targetsdownstream p53were stimulated, leading to the improvement of aging, as well as differentiation potential. And osteoporosis was significantly improved by apocynin invivo.Taken together, we determined several kinds of small-molecule compounds, whichhave a significant potential in improving aging process and treatment of age-related bonediseases, and provided a new train of thought in stem cell-based treatment of osteoporosis.