| With the development of aging population,transportation,sports and other undertakings in our country,the bone defects caused by trauma,infections,tumors,etc.increase year by year,and the clinical requirements for the treatment of bone defects are increasingly urgent.Bone defect treatment drugs mainly solve the two major problems of bone injury repair and prevention of postoperative infections.At present,although clinical medicines have good curative effect,they are restricted by factors such as price,side effects and single pharmacological effect.It requires a combination of multiple drugs,which will often increase the burden of patients and increase the risk of medication such as adverse reactions.If a drug can be found to repair bone defect and prevent postoperative infection,it will provide new ideas and methods for clinical treatment of bone tissue defect.Modern pharmacological studies have shown that the extracts of Chinese medicinal Semen strychni have analgesic,anti-inflammatory,antibacterial,anti-tumor and chondrocyte proliferation effects.Brucine as the main active ingredient of Semen strychni,can brucine be further resistant to clinical drug-resistant bacteria?can it also promote the proliferation of osteoblasts?The above assumptions have not been studied in depth.Therefore,this article aims to explore the antibacterial activity of brucine sulfate?BS?and its effect on osteoblast proliferation.The research content mainly includes the following two aspects:The first part,firstly,preparing BS through the steps of reflux,acid extraction,extraction,purification and crystallization,and then to perform qualitative and quantitative analysis on BS by high performance liquid chromatography?HPLC?.Secondly,the Oxford Cup method was used to initially investigate the growth inhibitory effect of BS on E.coli and S.aureus standard strains.Turbidimetric method to study the bacteriostatic concentration curve and time curve of BS against E.coli and S.aureus?standard strain and resistant strain?.At the same time,scanning electron microscopy?SEM?was used to observe the microscopic morphology of the bacteria treated with BS and to discuss its antibacterial mechanism.The conclusions of this study are listed as follows:?1?.BS was a white crystal.HPLC analysis showed that the retention time of the extracted BS was basically as the same as the standard brucine,and BS purity was 95.3%.?2?.Antibacterial results show that BS has a good antibacterial effect on E.coli and S.aureus?standard strain and drug resistant strain?,and it was the most sensitive to E.coli drug resistant strain.and the antibacterial activity after a single-dose was up to 3 days.?3?After bacteria were treated with BS,the surface morphology of E.coli and S.aureus bacteria changed,such as shrinkage,fusion and other phenomena.The antibacterial mechanism may be related to the destruction of bacterial cell wall.The second part,firstly,cell counting kit-8?CCK8?method was used to determine the half maximal inhibitory concentration(IC50)and proliferation effect of BS on hFOB1.19 osteoblasts.Subsequently,cellular immunofluorescence?IF?,Western blot?WB?and real-time fluorescence quantitative PCR?q PCR?experiments were performed,the mechanism of BS promoting the proliferation of hFOB1.19 human osteoblasts was discussed at the protein and gene levels.The conclusions of this study are listed as follows:?1?The CCK8 results showed that the IC50concentration of BS on hFOB1.19 osteoblasts was 0.23 mg/m L.When the concentration was0.08?g/m L?1?g/m L,it had a proliferation effect on hFOB1.19 osteoblasts.?2?BS can upregulate the expression of Runx2,Cyclin D1 and?-catenin in total protein and accumulate the?-catenin protein in nucleus.For another,BS could up-regulated the m RNA expression levels of?-catenin,Runx2 and Cyclin D1 in hFOB1.19 cells and down-regulated the m RNA expression levels of GSK 3?.These results suggest that BS can promote the proliferation of hFOB1.19 osteoblasts by affecting the Wnt/?-catenin signaling pathway. |
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