目的:探讨四妙汤改善类风湿关节炎(RA)模型大鼠的作用机制。方法:采用胶原诱导法(CIA)建立RA模型大鼠,灌胃四妙汤水煎液低、中、高剂量组(3.15 g/kg、6.3 g/kg、12.6 g/kg),维生素D组(25 μg/kg)。给药周期为28天,记录大鼠一般情况。苏...目的:探讨四妙汤改善类风湿关节炎(RA)模型大鼠的作用机制。方法:采用胶原诱导法(CIA)建立RA模型大鼠,灌胃四妙汤水煎液低、中、高剂量组(3.15 g/kg、6.3 g/kg、12.6 g/kg),维生素D组(25 μg/kg)。给药周期为28天,记录大鼠一般情况。苏木精–伊红(HE)染色法观察踝关节组织病理改变;酶联免疫吸附剂检测法(ELISA)检测大鼠血清白细胞介素-17 (IL-17)、白细胞介素-10 (IL-10)、转化生长因子-β (TGF-β)含量;实时荧光定量PCR法(RT-PCR)检测大鼠关节滑膜组织中维生素D受体(VDR)、1-α羟化酶(CYP27B1)、24-羟化酶(CYP24A1)、叉头/翼状螺旋转录因子3 (Foxp3)、维甲酸相关孤儿核受体γt (RORγt) mRNA表达。结果:与空白组相比,模型组血清IL-17含量水平显著上升(P 0.05),血清IL-10、TGF-β含量水平显著下降(P 0.05);关节滑膜CYP24A1、RORγt mRNA表达水平显著上升(P 0.05),VDR、CYP27B1、Foxp3 mRNA表达水平显著下降(P 0.05)。与模型组相比,维生素D组及四妙汤高剂量组血清IL-17水平显著下降(P 0.05),维生素D组及四妙汤中、高剂量组血清IL-10水平显著上升(P 0.05),维生素D组及四妙汤高剂量组血清TGF-β水平显著上升(P 0.05);维生素D组及四妙汤中、高剂量组关节滑膜VDR、CYP27B1、Foxp3 mRNA表达水平显著上升(P 0.05),维生素D组及四妙汤低、中、高剂量组关节滑膜CYP24A1 mRNA表达水平显著下降(P 0.05),维生素D组及四妙汤中、高剂量组关节滑膜RORγt mRNA表达水平显著下降(P 0.05)。结论:四妙汤通过调节关节滑膜组织中VDR、CYP27B1、CYP24A1、Foxp3、RORγt mRNA表达情况,改善血清IL-17、IL-10、TGF-β水平,发挥其干预RA的作用。Objective: To explore the mechanism of action of Simiao decoction in improving the rat model of RA. Methods: Rat models of rheumatoid arthritis (RA) were established by collagen-induced arthritis (CIA). The rats were then administered with low, medium and high doses of Simiao decoction (3.15 g/kg, 6.3 g/kg, 12.6 g/kg) and vitamin D (25 ng/kg) by gavage. The administration period was 28 days, and the general conditions of the rats were recorded. Observation of pathological changes in ankle joint tissues by hematoxylin-eosin (HE) staining method. The contents of interleukin-17 (IL-17), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) in the serum of rats were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of VDR, CYP27B1, CYP24A1, Foxp3, and RORγt in the synovial tissue of rat joints were detected by real-time fluorescence quantitative PCR (RT-PCR). Results: Compared with the control group, the serum IL-17 content level in the model group significantly increased (P 0.05), while the serum IL-10 and TGF-β content levels significantly decreased (P 0.05);the mRNA expression levels of CYP24A1 and RORγt in the synovial membrane significantly increased (P 0.05), and the mRNA expression levels of VDR, CYP27B1, and Foxp3 significantly decreased (P 0.05). Compared with the model group, the serum IL-17 level in the vitamin D group and the high-dose Simiao decoction group significantly decreased (P 0.05), the serum IL-10 level in the vitamin D group and the medium and high-dose Simiao decoction groups significantly increased (P 0.05), and the serum TGF-β level in the vitamin D group and the high-dose Simiao decoction group significantly increased (P 0.05);the mRNA expression levels of VDR, CYP27B1, and Foxp3 in the synovial membrane in the vitamin D group and the medium and high-dose Simiao decoction groups significantly increased (P 0.05), the mRNA expression level of CYP24A1 in the synovial membrane in the vitamin D group and the low, medium, and high-dose Simiao decoction groups significantly decreased (P 0.05), and the mRNA expression level of RORγt in the synovial membrane in the vitamin D group and the medium- and high-dose Simiao decoction groups significantly decreased (P 0.05). Conclusion: Simiao decoction exerts its intervention effect on RA by regulating the mRNA expression of VDR, CYP27B1, CYP24A1, Foxp3, and RORγt in the synovial membrane and improving the levels of serum IL-17, IL-10, and TGF-β.展开更多
【目的】维生素D3(vitamin D3,VD3)是一类重要的类固醇激素,参与调控动物体内多种生理活动,具有生物活性的VD3代谢物1,25(OH)2D3通过与维生素D受体(vitamin D receptor,VDR)结合发挥生物学功能。试验旨在克隆鹅VDR基因CDS序列并制备鹅VD...【目的】维生素D3(vitamin D3,VD3)是一类重要的类固醇激素,参与调控动物体内多种生理活动,具有生物活性的VD3代谢物1,25(OH)2D3通过与维生素D受体(vitamin D receptor,VDR)结合发挥生物学功能。试验旨在克隆鹅VDR基因CDS序列并制备鹅VDR多克隆抗体,为后续开展VDR介导VD3调控鹅体内多种生理活动的分子机制研究提供基础支撑。【方法】通过基因克隆方法获得鹅VDR基因CDS序列,并采用生物信息学方法对其编码蛋白进行结构和功能预测分析。通过基因合成和亚克隆方法构建鹅VDR基因重组表达质粒,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达和镍柱纯化后获得鹅VDR重组蛋白。以纯化的鹅VDR重组蛋白作为免疫原,制备兔抗鹅VDR多克隆抗体,采用间接ELISA法检测抗体效价,采用Western blotting检测抗体特异性。【结果】试验成功克隆了鹅VDR基因编码区序列,长度为1356 bp,编码451个氨基酸残基。该编码蛋白与鸡VDR蛋白的相似性高达91.4%,无信号肽和跨膜结构域,N-端含有核定位序列,且含有DNA结合域和配体结合域2个保守结构域,属于类固醇/甲状腺激素受体超家族成员。试验成功构建了pET-30a(+)-VDR重组质粒,并诱导表达鹅VDR重组蛋白。纯化后的重组蛋白纯度高达90%以上,且大小符合预期(52 ku)。试验制备了兔抗鹅VDR多克隆抗体,其抗体效价>1∶1093500;除肝脏组织外,该多克隆抗体在产蛋鹅肾脏、十二指肠、空肠和回肠组织中均能够识别天然VDR蛋白的2种亚型。【结论】试验成功克隆了鹅VDR基因CDS序列,并制备了兔抗鹅VDR多克隆抗体,该多克隆抗体特异性高,可用于后续鹅体内VDR介导的生物学功能研究。展开更多
文摘目的:探讨四妙汤改善类风湿关节炎(RA)模型大鼠的作用机制。方法:采用胶原诱导法(CIA)建立RA模型大鼠,灌胃四妙汤水煎液低、中、高剂量组(3.15 g/kg、6.3 g/kg、12.6 g/kg),维生素D组(25 μg/kg)。给药周期为28天,记录大鼠一般情况。苏木精–伊红(HE)染色法观察踝关节组织病理改变;酶联免疫吸附剂检测法(ELISA)检测大鼠血清白细胞介素-17 (IL-17)、白细胞介素-10 (IL-10)、转化生长因子-β (TGF-β)含量;实时荧光定量PCR法(RT-PCR)检测大鼠关节滑膜组织中维生素D受体(VDR)、1-α羟化酶(CYP27B1)、24-羟化酶(CYP24A1)、叉头/翼状螺旋转录因子3 (Foxp3)、维甲酸相关孤儿核受体γt (RORγt) mRNA表达。结果:与空白组相比,模型组血清IL-17含量水平显著上升(P 0.05),血清IL-10、TGF-β含量水平显著下降(P 0.05);关节滑膜CYP24A1、RORγt mRNA表达水平显著上升(P 0.05),VDR、CYP27B1、Foxp3 mRNA表达水平显著下降(P 0.05)。与模型组相比,维生素D组及四妙汤高剂量组血清IL-17水平显著下降(P 0.05),维生素D组及四妙汤中、高剂量组血清IL-10水平显著上升(P 0.05),维生素D组及四妙汤高剂量组血清TGF-β水平显著上升(P 0.05);维生素D组及四妙汤中、高剂量组关节滑膜VDR、CYP27B1、Foxp3 mRNA表达水平显著上升(P 0.05),维生素D组及四妙汤低、中、高剂量组关节滑膜CYP24A1 mRNA表达水平显著下降(P 0.05),维生素D组及四妙汤中、高剂量组关节滑膜RORγt mRNA表达水平显著下降(P 0.05)。结论:四妙汤通过调节关节滑膜组织中VDR、CYP27B1、CYP24A1、Foxp3、RORγt mRNA表达情况,改善血清IL-17、IL-10、TGF-β水平,发挥其干预RA的作用。Objective: To explore the mechanism of action of Simiao decoction in improving the rat model of RA. Methods: Rat models of rheumatoid arthritis (RA) were established by collagen-induced arthritis (CIA). The rats were then administered with low, medium and high doses of Simiao decoction (3.15 g/kg, 6.3 g/kg, 12.6 g/kg) and vitamin D (25 ng/kg) by gavage. The administration period was 28 days, and the general conditions of the rats were recorded. Observation of pathological changes in ankle joint tissues by hematoxylin-eosin (HE) staining method. The contents of interleukin-17 (IL-17), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) in the serum of rats were detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of VDR, CYP27B1, CYP24A1, Foxp3, and RORγt in the synovial tissue of rat joints were detected by real-time fluorescence quantitative PCR (RT-PCR). Results: Compared with the control group, the serum IL-17 content level in the model group significantly increased (P 0.05), while the serum IL-10 and TGF-β content levels significantly decreased (P 0.05);the mRNA expression levels of CYP24A1 and RORγt in the synovial membrane significantly increased (P 0.05), and the mRNA expression levels of VDR, CYP27B1, and Foxp3 significantly decreased (P 0.05). Compared with the model group, the serum IL-17 level in the vitamin D group and the high-dose Simiao decoction group significantly decreased (P 0.05), the serum IL-10 level in the vitamin D group and the medium and high-dose Simiao decoction groups significantly increased (P 0.05), and the serum TGF-β level in the vitamin D group and the high-dose Simiao decoction group significantly increased (P 0.05);the mRNA expression levels of VDR, CYP27B1, and Foxp3 in the synovial membrane in the vitamin D group and the medium and high-dose Simiao decoction groups significantly increased (P 0.05), the mRNA expression level of CYP24A1 in the synovial membrane in the vitamin D group and the low, medium, and high-dose Simiao decoction groups significantly decreased (P 0.05), and the mRNA expression level of RORγt in the synovial membrane in the vitamin D group and the medium- and high-dose Simiao decoction groups significantly decreased (P 0.05). Conclusion: Simiao decoction exerts its intervention effect on RA by regulating the mRNA expression of VDR, CYP27B1, CYP24A1, Foxp3, and RORγt in the synovial membrane and improving the levels of serum IL-17, IL-10, and TGF-β.
