摘要
目的探讨右归丸对膝骨关节炎(knee osteoarthritis,KOA)大鼠磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/核转录因子kappa B(nuclear factor kappa-B,NF-κB)信号通路的影响。方法SPF级SD大鼠60只,采用改良Hulth法复制膝骨关节炎大鼠模型,将大鼠分为假手术组,模型组,硫酸氨基葡萄糖组,右归丸高、中、低剂量组,每组10只,造模成功6周后给予药物干预,右归丸高、中、低剂量组按4.8、2.4、1.2 g/kg灌胃治疗,硫酸氨基葡萄糖组给予硫酸氨基葡萄糖0.17 g/kg灌胃治疗,假手术组和模型组给予等体积蒸馏水灌胃,干预8周后股动脉采血处死大鼠。HE法观察各组大鼠软骨组织病理改变并进行Mankin评分,免疫组化法检测各组大鼠软骨组织基质金属蛋白酶-14(matrix metalloproteinase-14,MMP-14)的蛋白表达,实时荧光定量PCR法检测各组大鼠软骨组织白细胞介素-6(interleukin-6,IL-6)、MMP-14 mRNA的基因表达水平,Western blot法检测各组大鼠软骨组织PI3K、磷酸化Akt(phosphorylated protein kinase B,pAkt)、MMP-14和NF-κB的蛋白表达。结果与假手术组相比,模型组大鼠Mankin评分(4.92±0.539)、IL-6(4.66±0.02)、MMP-14(2-ΔΔCt值5.70±0.02、评分结果4.27±0.15、蛋白灰度值0.89±0.03)、PI3K蛋白灰度值(1.19±0.09)、pAkt蛋白灰度值(2.48±0.22)、NF-κB(蛋白灰度值2.14±0.17)等指标均明显升高(P<0.01)。与模型组相比,右归丸高剂量组大鼠Mankin评分(3.05±0.253)、右归丸中、高剂量组MMP-14(2-ΔΔCt值3.47±0.05、1.08±0.01、评分结果3.07±0.37、0.76±0.12、蛋白灰度值0.59±0.03、0.28±0.07)和pAkt(蛋白灰度值1.51±0.13,1.30±0.07)等指标均明显降低,右归丸各干预组IL-6(2-ΔΔCt值2.27±0.09、1.65±0.05、1.15±0.04)、PI3K蛋白灰度值(0.67±0.11、0.62±0.11、0.45±0.05)和NF-κB蛋白灰度值(0.73±0.16、0.65±0.19、0.62±0.13)等指标均明显降低(P<0.05或P<0.01)。结论右归丸通过抑制PI3K/Akt/NF-κB信号通路中PI3K、pAkt、MMP-14和IL-6的表达达到治疗KOA的目的。
Objective To investigate the effects of Youguiwan on phosphatidyl inositol 3-kinase(PI3K)/protein Kinase B(Akt)/nuclear transcription factor Kappa B(NF-κB)signal pathway in knee osteoarthritis(KOA)rats induced by Hulth method.Methods Sixty SPF SD rats were randomly and equally divided into sham operation group,model group,Youguiwan high,medium,low dose group,glucosamine sulfate group(glucosamine sulfate),with 10 rats in each group.The modified Hulth method was used to make KOA models for 6 weeks.The rats were gavaged with corresponding drugs for 8 weeks.And HE staining was used to observe the morphological changes of articular cartilage of rats in each group.Immunohistochemistry was used to analyze the protein expression of matrix metalloproteinase-14(MMP-14).qRT-PCR was used to check the gene levels of IL-6 and MMP-14.Western blot was used to analyze the protein expressions of PI3K,pAkt,NF-κB.Results Compared with those of sham operation group,the levels of Mankin score(4.92±0.539),IL-6(4.66±0.02),MMP-14(2-ΔΔCt value 5.70±0.02,scoring results 4.27±0.15,protein gray value 0.89±0.03),PI3K(protein gray value 1.19±0.09),pAkt(protein gray value 2.48±0.22),NF-κB(protein gray value 2.14±0.17)were all obviously increased in model group(P<0.01).Compared with those of model group,the Mankin score(3.05±0.253)was declined in Youguiwan high-dose group,meatime,the levels of MMP-14(2-ΔΔCt value 3.47±0.05,1.08±0.01,scoring results 3.07±0.37,0.76±0.12,protein gray value 0.59±0.03,0.28±0.07)and pAkt(protein gray value 1.51±0.13,1.30±0.07)were also significantly reduced in Youguiwan middle-dose and high-dose group.The levels of IL-6(2-ΔΔCt value 2.27±0.09,1.65±0.05,1.15±0.04),PI3K(protein gray value 0.67±0.11,0.62±0.11,0.45±0.05)and NF-κB(protein gray value 0.73±0.16,0.65±0.19,0.62±0.13)were markedly decreased in Youguiwan intenvetion group(P<0.05).Conclusion It is effective in treating KOA induced Hulth method in rats,which is mainly achieved through inhibiting the expressions of IL-6,MMP-14,PI3K,pAkt and NF-κB by PI3K/Akt/NF-κB signaling pathway.
作者
安方玉
颜春鲁
张艳霞
金华
刘永琦
赵磊
夏鹏飞
成新平
靳安顺
杨晓蓉
AN Fang-yu;YAN Chun-lu;ZHANG Yan-xia;JIN Hua;LIU Yong-qi;ZHAO Lei;XIA Peng-fei;CHENG Xin-ping;JIN An-shun;YANG Xiao-rong(Teaching Experimental Training Center of Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Provincial-Level Key Laboratory for Molecular Medicine of Major Diseases and the Study on Prevention and Treatment of Traditional Chinese Medicine,Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Key Laboratory of Medicinal Chemistry and Quality Research,Colleges of Gansu Province,Lanzhou 730000,China;Key Laboratory of Dunhuang Medicine,Ministry of Education,Lanzhou 730000,China;Research and Test Center of Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;School of Public Health of Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;School of Basic Medical Science of Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China;Clinical Medical College of Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China)
出处
《中华骨质疏松和骨矿盐疾病杂志》
CSCD
北大核心
2019年第5期479-487,共9页
Chinese Journal Of Osteoporosis And Bone Mineral Research
基金
甘肃省高等学校科研项目(2018A-043)
甘肃省高等学校创新能力提升项目(2019A-080)
敦煌医学与转化省部共建教育部重点实验室开放基金项目(DHYX17-10)
甘肃省高校中(藏)药化学与质量研究省级重点实验室开放基金项目(zzy-2018-01)
甘肃省高校重大疾病分子医学与中医药防治研究重点实验室开放基金项目(FZYX17-18-10)
2018年度甘肃省高校大学生就业创业能力提升工程项目(6-1)
