云南白药对牙龈卟啉单胞菌诱导骨破坏的干预研究
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摘要
[目的]建立牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)感染的动物模型,客观评价云南白药对P.g诱导骨破坏的影响,为炎性骨病及牙周炎的预防、治疗提供新的思路。
[方法]将50日龄健康雄性昆明小鼠72只(体重20～25g),随机分为三组：模型组(M组=24只)、白药治疗组(Z=24只)、对照组(N组=24只)。模型组、白药治疗组在小鼠两耳中间颅顶至枕部骨的骨上,接种P. g1×108CFU/ml,200ul,分两次各日注射,对照组注射同等剂量的生理盐水。白药治疗组在建模的当天开始给予云南白药灌胃每只0.5ml(12.5mg/ml),模型组和对照组则给予同等剂量的生理盐水灌胃。建模的第5、8、14天分批各组各处死8只,运用HE染色病理检查各组的炎症情况及骨破坏情况；ELISA方法检测各组中血清TNF-α、IL-6的含量变化；TRAP染色法检测头颅骨中破骨细胞的含量；RT-PCR检测头颅骨中RANKLmRNA、OPGmRNA的表达,并对实验结果进行统计学分析。
[结果]比较第5、8、14天,白药治疗组、模型组和对照组小鼠血清中TNF-α、IL-6的含量、破骨细胞的数目及头颅骨中RANKL/GAPDH的比值,发现在第5天时各个指标在模型组中都是最高的,且白药治疗组<模型组有差异(p<0.05),两组和对照组相比数值均高有差异(p<0.05)。在各组之间进行OPG/GAPDH比值的比较中发现第5、8、14天白药治疗组都明显高于模型组和对照组有差异(p<0.05),且在第8天达到高峰；而模型组和对照组比较则在第5天时模型组低于对照组有差异(p<0.05),第8天时模型组高于对照组有差异(p<0.05),第14天高于对照组无差异(p>0.05)。
[结论]云南白药对抑制骨的破坏和提高骨的修复有明显的作用,可降低血液中IL-6、TNF-α的表达；下调RANKLmRNA的表达,上调OPGmRNA的表达；抑制破骨细胞的形成,从而抑制骨的吸收促进骨的修复。
Objective:To establish the experimental animal model that was infected with Porphyromonas gingivalis (P.g). Evaluation of effects of Yunnan Baiyao interfere P.g induces bone destruction.Searching for the new way to use in the prevention and treatment of inflammation disease of bone and periodontitis.
Methods:72 health male Kunming rats,50-day old,were randomly divided into three group:model group (group M=24)、treatment with Yunnan Baiyao group (group Z=24) and control group(group N=24).The rats of group M and Z had been inoculated with P.g (1*108CFU/ml) 200ul at the midline of the scalp between the ears twice daily; group N got the equal volumn saline with the same position and methods. At the same day the established of model, group Z had gotten the gastric perfusion Yunnan Baiyao 0.5ml(12.5mg/ml) for each rat daily,while group M and N just doing gastric perfusion with saline 0.5ml.8 rats on each group were killed on the day of 5th、8th and 14th. HE staining was employed to detect the inflammation condition and bone destroy. ELISA was employed to detect the level of TNF-α、IL-6 in serum.TRAP was employed to calculate the number of osteoclast in calvaria. RT-PCR was employed to detect the expression of RANKLmRNA and OPGmRNA in calvaria. Statistical analysis had been done at the end.
Result:Doing analysis of the level of TNF-a and IL-6、the number of osteoclast and the RANKLmRNA expression in the group M、Z and N on the day of 5th、8th and 14th. The data was apparently difference between group M and Z(p<0.05),and on the day of 5th the data was significant increase in group M;the data also demonstrated that group Z and M had increased contrast with group N (p<0.05).Doing analysis of the OPGmRNA expression in the group M、Z and N. The data in the group Z apparently higher than in the group M and N(p<0.05),and the biggest difference happened at the day of 8th.Compare group M with N,the data was lower in group M on the day of 5th(p<0.05),but it became higher than group N on the day of 5th(p<0.05) and 14th(p>0.05).
Discussion:The results demonstrated that Yunnan Baiyao is effective to inhibit bone destruction and improve bone repair. That include decrease the level of TNF-a and IL-6 in serum, decrease the expression of RANKLmRNA,enhance the expression of OPGmRNA and inhibit the production of osteoclast.
[方法]将50日龄健康雄性昆明小鼠72只(体重20～25g),随机分为三组：模型组(M组=24只)、白药治疗组(Z=24只)、对照组(N组=24只)。模型组、白药治疗组在小鼠两耳中间颅顶至枕部骨的骨上,接种P. g1×108CFU/ml,200ul,分两次各日注射,对照组注射同等剂量的生理盐水。白药治疗组在建模的当天开始给予云南白药灌胃每只0.5ml(12.5mg/ml),模型组和对照组则给予同等剂量的生理盐水灌胃。建模的第5、8、14天分批各组各处死8只,运用HE染色病理检查各组的炎症情况及骨破坏情况；ELISA方法检测各组中血清TNF-α、IL-6的含量变化；TRAP染色法检测头颅骨中破骨细胞的含量；RT-PCR检测头颅骨中RANKLmRNA、OPGmRNA的表达,并对实验结果进行统计学分析。
[结果]比较第5、8、14天,白药治疗组、模型组和对照组小鼠血清中TNF-α、IL-6的含量、破骨细胞的数目及头颅骨中RANKL/GAPDH的比值,发现在第5天时各个指标在模型组中都是最高的,且白药治疗组<模型组有差异(p<0.05),两组和对照组相比数值均高有差异(p<0.05)。在各组之间进行OPG/GAPDH比值的比较中发现第5、8、14天白药治疗组都明显高于模型组和对照组有差异(p<0.05),且在第8天达到高峰；而模型组和对照组比较则在第5天时模型组低于对照组有差异(p<0.05),第8天时模型组高于对照组有差异(p<0.05),第14天高于对照组无差异(p>0.05)。
[结论]云南白药对抑制骨的破坏和提高骨的修复有明显的作用,可降低血液中IL-6、TNF-α的表达；下调RANKLmRNA的表达,上调OPGmRNA的表达；抑制破骨细胞的形成,从而抑制骨的吸收促进骨的修复。
Objective:To establish the experimental animal model that was infected with Porphyromonas gingivalis (P.g). Evaluation of effects of Yunnan Baiyao interfere P.g induces bone destruction.Searching for the new way to use in the prevention and treatment of inflammation disease of bone and periodontitis.
Methods:72 health male Kunming rats,50-day old,were randomly divided into three group:model group (group M=24)、treatment with Yunnan Baiyao group (group Z=24) and control group(group N=24).The rats of group M and Z had been inoculated with P.g (1*108CFU/ml) 200ul at the midline of the scalp between the ears twice daily; group N got the equal volumn saline with the same position and methods. At the same day the established of model, group Z had gotten the gastric perfusion Yunnan Baiyao 0.5ml(12.5mg/ml) for each rat daily,while group M and N just doing gastric perfusion with saline 0.5ml.8 rats on each group were killed on the day of 5th、8th and 14th. HE staining was employed to detect the inflammation condition and bone destroy. ELISA was employed to detect the level of TNF-α、IL-6 in serum.TRAP was employed to calculate the number of osteoclast in calvaria. RT-PCR was employed to detect the expression of RANKLmRNA and OPGmRNA in calvaria. Statistical analysis had been done at the end.
Result:Doing analysis of the level of TNF-a and IL-6、the number of osteoclast and the RANKLmRNA expression in the group M、Z and N on the day of 5th、8th and 14th. The data was apparently difference between group M and Z(p<0.05),and on the day of 5th the data was significant increase in group M;the data also demonstrated that group Z and M had increased contrast with group N (p<0.05).Doing analysis of the OPGmRNA expression in the group M、Z and N. The data in the group Z apparently higher than in the group M and N(p<0.05),and the biggest difference happened at the day of 8th.Compare group M with N,the data was lower in group M on the day of 5th(p<0.05),but it became higher than group N on the day of 5th(p<0.05) and 14th(p>0.05).
Discussion:The results demonstrated that Yunnan Baiyao is effective to inhibit bone destruction and improve bone repair. That include decrease the level of TNF-a and IL-6 in serum, decrease the expression of RANKLmRNA,enhance the expression of OPGmRNA and inhibit the production of osteoclast.
引文
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