| BackgroundOsteoporosis is a skeletal disorder characterized by decreased bone mass and damage bone tissue microstructure, culminating in fragility fractures, pain and disability. As the incidence of osteoporosis has been increasing with age, it has significantly increased the social burden and affected quality of life of elders with aging society.The main pathological process of osteoporosis is an imbalance between bone resorption and bone formation, but mechanisms in detail are still unclear. Resent years there is growing evidence that the level of serum peroxide in patients with osteoporosis increased while antioxidant decreased compared with controls, which gets attention to the role of oxidative stress in pathogenesis of osteoporosis. Previous studies indicate that in the development of osteoporosis oxidative stress may affect bone metabolism through consuming antioxidant, activating osteoclasts and inhibiting osteoblasts and bone formation.Oxidative stress is an imbalance between the free radicals and antioxidant mechanism in biological systems and can make an accumulation of reactive oxygen species (ROS) in the body. At present, the most common markers of oxidative stress are protein carbonyl, lipid peroxide and advanced glycation end products (AGEs). Because protein carbonyl detection occurred earlier than lipid peroxidation and the possibility of direct interaction between hydroxyl radical and protein is20times higher than with DNA, it is generally believed that protein is the main original target of ROS. Therefore, clarifying the role of oxidative modification of protein and its products in human diseases will provide new theories for researches of oxidative stress related diseases, and new ways or targets for intervention.Oxidative protein products retention has involved in many important chronic diseases including diabetes, metabolic syndrome, chronic kidney disease and atherosclerosis. It has been demonstrated that some modificative protein products such as AGEs are related to many chronic diseases containing dialysis-related amyloidosis, Alzheimer disease, and diabetic microangiopathy and diabetic osteoporosis. All these studies have demonstrated that oxidative protein products retention is not only the result/marker of oxidative stress, but also disease-causing agents involved in the development of many important human deseases.Protein quantity in human body is huge, and albumin which is most susceptible to oxidative modification is also the main protein in the cycle. Advanced oxidation protein products are a kind of protein conjugate containing tyrosine by chlorination and oxidation reaction with serum albumin. They are first discovered in the plasma of patients with uremia. Recent studies suggest that AOPPs, a kind of uremic toxin causing inflammatory, may not only be the product of oxidative stress, but also could induce or aggravate oxidative stress. It has been demonstrated that AOPPs involve in obesity, renal fibrosis, aging, age-related cataract, and so on. Osteoporosis is also an age-related disease. It has been indicated that the level of AOPPs in patients with osteoporosis increases compared with control. Oxidative stress can also raise AOPPs in MC3T3-E1cells, but the mechanisms in detail between AOPPs and osteoporosis are still unclear.Receptor for advanced glycation end products (RAGE) is multiple ligand receptor in mmunoglobulin superfamily that can combine with advanced glycation end products (AGEs) and AOPPs. The level of AOPPs is usually low in human, but rises in oxidative stress such as inflammation and diabetes following RAGE expression increased dramatically. Experiments using RAGE knockout mice show an increased bone mass and bone biomechanical strength and a decreased number of osteoclasts in RAGE-/-mice compared to wild-type mice; RAGE overexpression by lenti virus transfection inhibits osteoblast proliferation via suppression of Wnt, PI3K and ERK pathways; AGEs-RAGE interaction can induce the generation of ROS through NADPH oxidase resulting in apoptosis of osteoblasts/MSCs, and inhibition of proliferation and differentiation of osteoblasts/MSCs. The studies above all indicate that RAGE plays a modulatory role in development of osteoporosis. The biological structure and activity of AOPPs are similar as AGEs. Combining previous researches about AGEs, we speculate that AOPPs might inhibit the proliferation and osteogenic differentiation of MSCs/OB by increasing ROS generation following an up-regulating of RAGE expression.As bone formation is regulated by osteoblasts which originate from MSCs, the negative effects on MSCs/OB osteogenic differentiation may aggravate the development of osteoporosis. So in this research we induced rat MSCs and OB with different concentrations of AOPPs, and detected indexes about proliferation, differentiation, ROS and expression of RAGE in MSCs/OB to determine the effect of AOPPs on osteoporosis.ObjectivesIn this project we aimed to establish the injury model by treating rat MSCs and osteoblasts with AOPPs. We detected the indexes about proliferation and osteogenic differentiation of rat MSCs and osteoblasts, and tested the levels of ROS and RAGE expression, to discuss the effects and mechanisms of AOPPs on MSCs and osteoblasts.ContentThe whole project includes two parts: Part I The effects and mechanisms of AOPPs on proliferation and osteogenic differentiation of rat MSCs Section I The effects of AOPPs on proliferation and osteogenic differentiation of rat MSCsObjectivesWe aimed to establish the injury model by treating rat MSCs with AOPPs, and to analyze the effects of AOPPs on proliferation and osteogenic differentiation of MSCs.Methods1. Isolation, culture and identification of cellsMSCs were isolated from SD rat’s bone marrow from femurs and were cultured with FBS-DMEM medium at37℃in a humidified atmosphere of5%CO2. These cells were identified for surface marker by flow cytometry and for the adipogenic and osteogenic differentiation capability by oil red O staining and alizarin red staining.2. Preparation of AOPPs in vitroBriefly, BSA was dissolved in phosphate-buffered saline with HOCl, and incubated for30min in the dark. AOPPs was identified using a spectrophotometer at340nm.3. Assessment of cell proliferation by colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-dihenyltetrazolium bromide (MTT) assayMSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h (In this manuscript, we grouped according to different concentrations and time); cells were cultrured by200μg/ml AOPPs alone for0,24,48,72h respectively.The cells are planted in96-well plates. After each experimental treatment, add MTT solution to each hole, and add150μl DMSO to each hole. Absorption was measured at570nm with a microplate reader. The absorbance measured by MTT assay reflects cells injury.4. Assessment of ALP activity by P-nitrophenyl Phosphate (PNPP) assayMSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h; cells were cultrured by200μg/ml AOPPs alone for0,24,48,72h respectively. Cells were lysed by0.1%TritonX-100, then detected ALP activity by PNPP assay, and tested protein concentrations by BCA assay.5. Detection of calcified nodules by alizarin red stainingCells were treated by AOPPs at concentrations of50,100,200,400μg/ml for1week after being cultured in osteogenic differentiation medium for2weeks. Then cells were stained using alizarin red to detect calcified nodules formation.6. Detection of ALP and collagen I mRNA levels by RT-PCR After being induced with osteogenic differentiation medium for1week, MSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h. Then total RNA was extracted using Trizol, and was detected by RT-PCR.Statistical AnalysisAll analyses were carried out with SPSS18.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p <0.05.Results1. MSCs were isolated from rat femur bone marrow and formed elongated spindle or polygon. The surface marker CD34was negative while CD29was positive. After induced by adipogenic differentiation and osteogenic differentiation medium, lipid droplets in cells and bone nodules were observed respectively.2. After treatment with50,100,200,400∴g/ml AOPPs for72h, the OD value were0.4722±0.0050,0.4563±0.0170,0.4170±0.0147,0.3770±0.0266, respectively, and significantly lower than control group (0.5397±0.0080) and BSA group (0.5273±0.0297)(P=0.000).3. Comparison OD value of cells after being incubated with AOPPs for different time showed that a significant negative correlation exists between the OD value of MSCs and time (0,24,48,72h)(P=0.000).4. After treatment with50,100,200,400μg/ml AOPPs for72h, the ALP activities (U/g-protein) were92.9291±4.2248,87.4805±3.8420,85.2812±1.5028, 81.6935±2.0550, respectively, and significantly lower than control group (98.1610±2.0889) and BSA group (97.4307±3.5394)(P<0.05).5. Comparison ALP activities of cells after being incubated with AOPPs for different time showed that a significant negative correlation exists between the ALP activities of MSCs and time (0,24,48,72h)(P=0.000)6. After incubated with AOPPs, red calcified nodules were observed in control group and BSA group, but few in AOPPs group.7. After treatment with50,100,200,400μg/ml AOPPs for72h, the ALP mRNA relative levels were0.4557±0.0661,0.2945±0.0464,0.2182±0.0311,0.1632±0.0379, respectively, and significantly lower than BSA group (0.9058±0.0876)(P=0.000). The Collagen I mRNA relative levels were0.5575±0.0276,0.4755±0.0457,0.3827±0.0507,0.1893±0.0219, respectively, and significantly lower than BSA group (0.9123±0.0108)(P=0.000).ConclusionThe cells isolated were identified as MSCs. The inhibition of proliferation and ALP activities were induced by AOPPs in a dose, time-dependent manner. AOPPs could also down-regulate ALP and Collagen I mRNA levels, and inhibit the calcified nodules formation. All the results indicated that AOPPs could inhibit osteogenic differentiation of MSCs.Section Ⅱ The effects of AOPPs on oxidative stress and RAGE expression in MSCsObjectivesWe analyzed the level of oxidative stress by fluorescence microscop, and tested the mean fluorescence intensity (MFI) by flow cytometry. RAGE mRNA and protein levels were detected using RT-PCR and western blot respectively. All were to discuss the mechanism of AOPPs on MSCs.Methods1. Detection of reactive oxygen species (ROS) MSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for2h. The level of ROS was quantified by2’,7’-dichlorofluorescein diacetate assay using fluorescence microscop and flow cytometry.2. Detection of RAGE mRNA level by RT-PCRMSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h. Then total RNA was extracted using Trizol, and was detected by RT-PCR.3. Detection of RAGE protein level by western blotCells with various treatments were collected and lysed in protein lysis buffer. The supernatant contained the cell extracts and the protein concentration was measured using the BCA protein assay. Equal amounts of protein (25μg) from each sample were separated on SDS-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in blocking buffer.for4h and incubated with anti-RAGE (1:500) and anti-GAPDH (1:500) antibody for4h. Secondary specific horseradish peroxidase-linked antibodies (1:5000,1:4000) were added for50minh, and immune complexes were detected by ECL chemiluminescence, the band intensity was measured and quantified with BIO-RAD software.Statistical AnalysisAll analyses were carried out with SPSS18.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p <0.05.Results1. Compared with control group, DCF fluorescence in cells exposed to AOPPs was strikingly increased by fluorescence microscop. The ratio of MFI in different concentration groups and the control group were175.7478±18.0156,328.8604±15.8096,441.6609±17.5993,579.6729±26.2623, respectively, and significantly higher than the ratio of BSA and control (111.4910±4.3167)(P=0.000).2. After treatment with50,100,200,400μg/ml AOPPs for72h, the RAGE mRNA relative levels were1.6144±0.2747,3.0609±0.4656,5.2401±0.3257, 5.6851±1.0159, respectively, and significantly higher than BSA group (1.2209±0.1104)(P=0.000) except50μg/ml AOPPs.3. After treatment with50,100,200,400μg/ml AOPPs for72h, the RAGE protein relative levels were0.3980±0.0145,0.4566±0.0122,0.5398±0.0208,0.6249±0.0246, respectively, and significantly higher than control group (0.3799±0.0249) and BSA group (0.3898±0.0209)(P<0.01) except50μg/ml AOPPs.ConclusionExposure to AOPPs for2h caused a significant increase in ROS generation, and AOPPs also increased RAGE expression in both mRNA and protein levels. All these indicate that AOPPs may increase the RAGE expression after combined with RAGE, meanwhile increase the ROS level in MSCs, so that AOPPs could inhibit MSCs proliferation and osteogenic differention.Part Ⅱ The effects and mechanisms of AOPPs on proliferation and osteogenic differentiation of rat OBSection I The effects of AOPPs on proliferation and osteogenic differentiation of rat OBObjectivesWe aimed to establish the injury model by treating rat OB with AOPPs, and to analyze the effects of AOPPs on proliferation and osteogenic differentiation of OB.Methods1. Isolation, culture and identification of cellsOB were isolated from newborn SD rat’s skull and were cultured with osteogenic differentiation medium at37℃in a humidified atmosphere of5%CO2. These cells were identified using alkaline phosphatase assay.2. MTT assayOB were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h; cells were cultrured by200μg/ml AOPPs alone for0,24,48,72h respectively.The cells are planted in96-well plates. After each experimental treatment, add MTT solution to each hole, and add150μl DMSO to each hole. Absorption was measured at570nm with a microplate reader. The absorbance measured by MTT assay reflects cells injury.3. Assessment of ALP activity by P-nitrophenyl Phosphate (PNPP) assayOB were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h; cells were cultrured by200μg/ml AOPPs alone for0,24,48,72h respectively. ALP activity was measured with the instruction of ALP kit.4. Detection of ALP and collagen I mRNA levels by RT-PCR OB were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h. Then total RNA was extracted using Trizol, and was detected by RT-PCR.Statistical AnalysisAll analyses were carried out with SPSS18.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p <0.05.Results1. OB were isolated from newborn SD rat’s skull and formed fusiform or polygon. The ALP staining was positive, and the red brown granules were observed in cytoplasm.2. After treatment with50,100,200,400μg/ml AOPPs for72h, the OD value were0.5207±0.0431,0.4947±0.0164,0.4087±0.0170,0.2673±0.0031, respectively, and significantly lower than control group (0.6290±0.0092) and BSA group (0.6170±0.0168)(P=0.000).3. Comparison OD value of cells after being incubated with AOPPs for different time showed that a significant negative correlation exists between the OD value of OB and time (0,24,48,72h)(P=0.000).4. After treatment with50,100,200,400μg/ml AOPPs for72h, the ALP activities (U/L) were40.1066±0.7870,37.0065±1.4205,31.2424±0.930520.5793±0.7350, respectively, and significantly lower than control group (53.2817±3.2130) and BSA group (52.3129±3.7520)(P=0.000). 5. Comparison ALP activities of cells after being incubated with AOPPs for different time showed that a significant negative correlation exists between the ALP activities of OB and time (0,24,48,72h)(P=0.000)6. After treatment with50,100,200,400μg/ml AOPPs for72h, the ALP mRNA relative levels were0.8960±0.0187,0.7017±0.0438,0.6044±0.0299,0.4765±0.0323, respectively, and significantly lower than BSA group (0.9648±0.0366)(P<0.05). The Collagen I mRNA relative levels were0.8811±0.0007,0.8151±0.0075,0.6378±0.0309,0.5730±0.0061, respectively, and significantly lower than BSA group (0.9664±0.0113)(P=0.000).ConclusionThe cells isolated were identified as OB. The inhibition of proliferation and ALP activities were induced by AOPPs in a dose, time-dependent manner. AOPPs could also down-regulate ALP and Collagen I mRNA levels wich indicated that AOPPs could inhibit osteogenic differentiation of OB.Section II The effects of AOPPs on oxidative stress and RAGE expression in OBObjectivesWe analyzed the level of oxidative stress by fluorescence microscop, and tested the mean fluorescence intensity (MFI) by flow cytometry. RAGE mRNA level was detected using RT-PCR. All were to discuss the mechanism of AOPPs on OB.Methods1. Detection of reactive oxygen species (ROS) Use the same method with that of Part Ⅰ-SectionⅡ.2. Detection of RAGE mRNA level by RT-PCR Use the same method with that of Part Ⅰ-Section Ⅱ.Statistical AnalysisAll analyses were carried out with SPSS18.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p <0.05.Results1. Compared with control group, DCF fluorescence in cells exposed to AOPPs was strikingly increased by fluorescence microscop. The ratio of MFI in different concentration groups and the control group were183.6783±14.1578,307.5650±13.8079,366.9337±20.0321,606.2912±46.2415, respectively, and significantly higher than the ratio of BSA and control (112.7752±6.7175)(P<0.01).2. After treatment with50,100,200,400μg/ml AOPPs for72h, the RAGE mRNA relative levels were1.3626±0.1090,2.9922±0.3198,5.1792±0.3936,5.8637±0.3561, respectively, and significantly higher than BSA group (1.0703±0.0557)(P=0.000) except50μg/ml AOPPs.ConclusionExposure to AOPPs for2h caused a significant increase in ROS generation, and AOPPs also increased RAGE expression in mRNA level. All these indicate that AOPPs may increase the RAGE expression after combined with RAGE, meanwhile increase the ROS level in OB, so that AOPPs could inhibit OB proliferation and osteogenic differention. |
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