| Objective To investigate the protection of puerarin on osteoblasts(OB)by observing the effects of puerarin and H2O2 on the proliferation,differentiation and mineralization function of OB damaged by H2O2 in vitro.Methods①The crania of Wistar rats which were born within 24 hours were obtained in aseptic condition.After enzyme digestion and isolation repeatedly,purified OB were cultured for further study.We observed osteoblasts' morphologic character in the phase-contrast microscopy.Osteoblasts were identified by cytochemical staining of alkaline phosphatase(ALP),immunohistochemistry staining of osteocalcin(OCN)and other methods.The passage 2 osteoblast cells were used in experiment.②OB were randomly divided into three groups:the normal control group,various concentrations of puerarin(10-10～10-3mol/L,'P')groups and H2O2(10-9～10-3mol/L,'H')groups.After several days culture in different mediums,the following study were continued:The OD values on proliferation of OB(from the 1stto 4thdays)and expression of ALP(from the 2ndto the 8thdays after induction and culture)in each group were determined by microplate reader.The mineral nodes of OB(when were induced-cultured two weeks) were dyed in alizarin red and counted.③Osteoblasts were treated with 'P' 48 hours,and meanwhile treated with 0.1mmol/L H2O2('H′')24 hours[P+H′]and treated with 'H′' 24 hours,then incubated with 'P' 48 hours[H′+P].Then the proliferation and differentiation in each group were determined by MTT assay and activity of ALP.④The OB were treated with 10-5mol/L puerarin('P＇')48 hours,then treated with 'H′' 24 hours at the same time[P′+ H′],the content of malondialdehyde(MDA),the activity of superoxide dismutase (SOD)and the total antioxidative capability(T-AOC)were tested and compared.Results①Comparing with the normal control group in the proliferation of OB (P＜0.01),the expression of ALP(P＜0.01)and the number of mineral nodes of OB (P＜0.05),we found they have significantly increased in 'P' groups at the concentrations of (10-8～10-5)mol/L,especially in the concentration of 10-6mol/L(P＜0.01),we also found that puerarin increased the proliferation of OB from the 1stto the 4thdays and the maxiumum effect was on the 3rdday(P＜0.01).Puerarin also increased the differentiation of OB on the 2ndand the 4thdays(P＜0.01).At the concentration of 10-4mol/L,puerarin improved the proliferation and differentiation of OB on the 2ndand 4thdays respectively (P＜0.01),the numbers of mineral nodes of OB have no increase(P＞0.05).However, 10-3mol/L puerarin decreased the activity of ALP(P＜0.01)and the formation of mineral nodes(P＜0.05).②Comparing with the normal control group in MTT value(P＜0.05)and ALP activity(P＜0.001),we noticed dramatically raise at the lower concentration (1×10-8～6×10-5mol/L H2O2)in 'H' groups,MTT value and ALP activity were markedly reduced at the higher concentration(1×10-4～1×10-3mol/L H2O2)(P＜0.001),and the maxiumum effect was in the 'H′' group(P＜0.001).The effects of H2O2 on OB were always existed among the every experimental time,however the maximum effect was observed at the 3rdand the 6thdays respectively(P＜0.001).③Compared with the 'H′' group in MTT value(P＜0.01)and ALP activity(P＜0.001),we found markedly increase at the concentration(10-7～10-4mol/L puerarin)and the concentration(10-8～10-4mol/L puerarin) respectively,in 'P+H′' and 'H′+P' groups.The maximum effect was observed at 10-5mol/L puerarin group(P＜0.001).④Compared with the normal control group,the 'H′' group showed significantly increase in the content of mMDA,but markedly decrease at the activity of SOD and T-AOC(P＜0.01).Compared with the 'H′' group,puerarin made the mMDA of damaged OB significantly decrease(P＜0.01),the activicty of SOD(P＜0.01) and T-AOC(P＜0.01)were increase significantly.However,compared with the normal control group,puerarin still not make the mMDA(P＜0.01),the activiy of SOD(P＞0.05) and T-AOC(P＜0.01)of damaged OB recovered to the normal level.Conclusion①The two-way operating characteristics of puerarin and H2O2 existed in the proliferation and differentiation of OB.②Puerarin can prevent OB from oxidative damage induced by H2O2 in vitro. |
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