三叶人字草对IgA肾病的治疗作用及其机制研究
摘要
目的：
通过复制IgA肾病小鼠模型,观察邓老验方中三叶人字草对IgA肾病模型小鼠肾皮质血小板衍化生长因子(PDGF-B)及其mRNA表达的影响,测定IgA肾病模型小鼠血清中肌酐(SCR)、尿素氮(BUN)、一氧化氮(NO)水平,探讨其治疗IgA肾病的作用机理,并对三叶人字草治疗IgA肾病的相关药效学实验进行探索,观察其对免疫系统、血压、血液功能、肠胃功能四个方面的影响。为临床治疗IgA肾病提供实验研究依据,提升中医药在此领域的研究水平,为探索中医药高效低毒治疗方案提供新思路。
方法：
采用牛血清白蛋白(BSA)、葡萄球菌肠毒素B (SEB)成功复制IgA肾病小鼠模型,通过观察对模型小鼠的一般情况、测定蛋白尿、血尿量及血清中SCR, BUN、NO水平；半定量RT-PCR测定IgA肾病小鼠肾皮质PDGF-B、PDGFR-B mRNA的表达；并观察其肾脏病理组织切片,考察三叶人字草IgA肾病模型小鼠的作用机制。
采用碳粒廓清法,考察人字草对小鼠免疫系统的影响；采用大鼠灌服三叶人字草提取液,考察其对血压的影响；采用玻片法、毛细管法,考察三叶人字对小鼠凝血、出血时间以及其对血小板聚集等影响；采用半固体灌胃法,考察三叶人字草对小鼠胃肠功能的作用,探讨其治疗IgA肾病的相关药效学作用。
以上实验的供试药品三叶人字草水提液采用正交试验法,以总黄酮含量为指标,考察不同影响因素和水平对提取工艺条件的影响,筛选出最优工艺。
结果：
(1)三叶人字草对IgA肾病模型小鼠的药理作用及机制：
三叶人字草作用于IgA肾病小鼠后,能明显改善模型小鼠的生活状态,减轻IgA肾病模型小鼠的蛋白尿和尿血量,三叶人字草高剂量组(12.34 g／kg)能降低IgA肾病小鼠血清中SCR、BUN含量,提高IgA肾病小鼠全血中NO活力(P<0.05),能降低模型小鼠肾组织PDGF-B、PDGFR-B mRNA的表达。IgA肾病模型小鼠PDGF-B、PDGFR-B基因RT-PCR产物进行琼脂糖凝胶电泳结果显示：各组均在193bp处可见PDGF-B mRNA的表达,相比模型组,各组条带逐渐变暗,半定量分析提示三叶人字草高剂量组PDGF-B mRNA的表达下降26.8%,与模型组相比,具有显著性差异(P<0.05)；各组均在206bp处可见PDGFR-B mRNA的表达,相比模型组,各组条带逐渐变暗,半定量分析提示三叶人字草高剂量组PDGF-B mRNA的表达下降18.5%,与模型组相比,具有显著性差异(P<0.05)。
(2)三叶人字草治疗IgA肾病的相关药效学研究：
高剂量(12.34 g／kg)、中剂量(6.17 g／k (3)正交实验筛选出三叶人字草最佳提取工艺为：以水为提取溶剂,16倍料液比,煎煮0.5h,提取3次。
结论：
三叶人字草能减少IgA肾病小鼠尿蛋白和血尿,降低IgA肾病小鼠血清中BUN、SCR含量,改善IgAN症状；能显著提高小鼠体内NO活力,能降低IgA肾病小鼠肾皮质中PDGF-B、PDGFR-B mRNA的表达,减少因PDGF-B及受体持续过度表达刺激系膜细胞增生及基质积累而导致肾脏疾病进行性发展,并能增强免疫,降压,止血,对胃肠功能有促进作用。三叶人字草对IgAN模型小鼠具有治疗作用,其表现出的肾脏保护效应,可能与抑制肾组织中PDGF-B、PDGFR-B mRNA的表达有重要作用,为其治疗IgA肾病的机制之一。
Objective:
Observe the effect of Kummerowia Striata from Denglao experience prescription on the expression of platelet-derived growth factor (PDGF-B) and its mRNA in IgA nephropathy model mouse. Determine the serum level of SCR、BUN、NO in IgA nephropathy model mice. Study the Kummerowia Striata's treatment on IgA nephropathy and the IgA nephropathy mechanism of pathogenesis. Besides, explore the pharmacodynamics of Kummerowia Striata treatment on IgAN and observe its effect on the immune system、blood pressure、blood function and gastrointestinal function etc. Provide experimental foundation for the clinical treatment of IgAN and promote the research level of traditional Chinese medicine in the nephritis research area. Advance the treatment of low toxicity and efficient of traditional Chinese medicine and provide a new method of treatment.
Method:
The extraction conditions were optimized by orthogonal test method. Investigated by orthogonal test method and level of different factors on the extraction rate, Preparation of this selection process with the Kummerowia Striata extract as the experimental drugs tested.
Using bovine serum albumin (BSA) and staphylococcal enterotoxin B (SEB) replicate IgA nephropathy mouse model. Observe the general situation of mice, serum SCR, BUN, NO level; determinate the expression of PDGF-B, PDGFR-B mRNA in IgA nephropathy mouse renal cortex by Semi-quantitative RT-PCR. Study the renal histopathology of Kummerowia Striata's treatment on IgA nephropathy model mice.
Effect of Kummerowia Striata on the immune system in normal mice was used by carbon clearance method. To study its effects on blood pressure by slide method. Study trefoil clotting and bleeding time and its platelet aggregation and other hemostatic factor by capillary method. Study the effects on gastrointestinal function to promote its treatment on IgA nephropathy pharmacodynamic by semi-solid method.
Results:
(1) pharmacological effects of Kummerowia Striata on IgA nephropathy model mice and its mechanism
As the Kummerowia Striata effect on IgA nephropathy model mice, the living conditions of mice significantly improved while proteinuria and the amout of hematuria reduced. The content serum of SCR, BUN decreased and the whole blood NO activity (P<0.05) increased in the high dose group(12.34 g/kg).The expression of mice renal cortex PDGF-B、PDGFR-B mRNA also reduced in the high dose group. Agarose gel eletrophoresis of products from PDGF-B、PDGFR-B mRNA gene RT-PCR of IgAN model mice showed that:193bp in each group were observed at the expression of PDGF-B mRNA,the other groups gradually darkened strip compared with model group. Semi-quantitative analysis showed that the expression of PDGF-B mRNA decreased 26.8% in KS high dose group with significant differences (P<0.05) compared with model group; 203bp in each group were observed at the expression of PDGFR-B mRNA, the other groups gradually darkened strip compared with model group. Semi-quantitative analysis showed that the expression of PDGF-B mRNA decreased 18.5% in KS high dose group with significant differences(P<0.05) compared with model group.
(2) Effect of Kummerowia Striata on IgA nephropathy model mice and its pharmacodynamics research
The mice peritoneal macrophage phagocytic clearance capacity and speed were significantly increased(P<0.01) in KS high dose group (12.34 g/kg) and medium dose group (6.17g/kg). The spleen of mice were significantly improved in KS high dose group and medium dose group. KS high dose could increase the normal mouse peritoneal macrophage phagocytosis (P<0.01). KS high、medium and low dose might have an effect on the rats blood pressure decreasing but have no significant difference compared with the former administration. KS high and medium dose could significantly shorten clotting time (slide method, capillary method) (P<0.01); KS high dose group mice have significantly effect of shorten the bleeding time (P<0.01) and increase platelet count (PLT) (P<0.01). But on the red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT) have no obvious indicators of the role of trend. KS high dose could significantly promote gastric emptying (P<0.01) and intestinal propulsiving (P<0.01).
(3)The best extraction of orthogonal filtering out KS:Water as extraction solvent,16 times the ratio of solid to liquid, boiling 0.5h, extract 3 times.
Conclusion:
KS could reduce proteinuria and the amout of hematuria in IgAN model mice, lower serum content of BUN, SCR and heal the symptoms of IgAN; significantly increase NO activity and reduce the renal cortex expression of PDGF-B and PDGFR-B mRNA in IgAN model mice;reduce the development of renal disease which is due to PDGF-B and its receptor stimulation sustained progressively over-expression of mesangial cell proliferation and matrix accumulation. KS also can enhance immunization, blood pressure, bleeding, promote gastrointestinal function. The protective effect of KS on IgAN model mice could be interrelated with the expression of PDGF-B and PDGFR-B mRNA that they might play an important role in IgAN and that may be one of the mechanisms of KS effect on IgAN.
通过复制IgA肾病小鼠模型,观察邓老验方中三叶人字草对IgA肾病模型小鼠肾皮质血小板衍化生长因子(PDGF-B)及其mRNA表达的影响,测定IgA肾病模型小鼠血清中肌酐(SCR)、尿素氮(BUN)、一氧化氮(NO)水平,探讨其治疗IgA肾病的作用机理,并对三叶人字草治疗IgA肾病的相关药效学实验进行探索,观察其对免疫系统、血压、血液功能、肠胃功能四个方面的影响。为临床治疗IgA肾病提供实验研究依据,提升中医药在此领域的研究水平,为探索中医药高效低毒治疗方案提供新思路。
方法：
采用牛血清白蛋白(BSA)、葡萄球菌肠毒素B (SEB)成功复制IgA肾病小鼠模型,通过观察对模型小鼠的一般情况、测定蛋白尿、血尿量及血清中SCR, BUN、NO水平；半定量RT-PCR测定IgA肾病小鼠肾皮质PDGF-B、PDGFR-B mRNA的表达；并观察其肾脏病理组织切片,考察三叶人字草IgA肾病模型小鼠的作用机制。
采用碳粒廓清法,考察人字草对小鼠免疫系统的影响；采用大鼠灌服三叶人字草提取液,考察其对血压的影响；采用玻片法、毛细管法,考察三叶人字对小鼠凝血、出血时间以及其对血小板聚集等影响；采用半固体灌胃法,考察三叶人字草对小鼠胃肠功能的作用,探讨其治疗IgA肾病的相关药效学作用。
以上实验的供试药品三叶人字草水提液采用正交试验法,以总黄酮含量为指标,考察不同影响因素和水平对提取工艺条件的影响,筛选出最优工艺。
结果：
(1)三叶人字草对IgA肾病模型小鼠的药理作用及机制：
三叶人字草作用于IgA肾病小鼠后,能明显改善模型小鼠的生活状态,减轻IgA肾病模型小鼠的蛋白尿和尿血量,三叶人字草高剂量组(12.34 g／kg)能降低IgA肾病小鼠血清中SCR、BUN含量,提高IgA肾病小鼠全血中NO活力(P<0.05),能降低模型小鼠肾组织PDGF-B、PDGFR-B mRNA的表达。IgA肾病模型小鼠PDGF-B、PDGFR-B基因RT-PCR产物进行琼脂糖凝胶电泳结果显示：各组均在193bp处可见PDGF-B mRNA的表达,相比模型组,各组条带逐渐变暗,半定量分析提示三叶人字草高剂量组PDGF-B mRNA的表达下降26.8%,与模型组相比,具有显著性差异(P<0.05)；各组均在206bp处可见PDGFR-B mRNA的表达,相比模型组,各组条带逐渐变暗,半定量分析提示三叶人字草高剂量组PDGF-B mRNA的表达下降18.5%,与模型组相比,具有显著性差异(P<0.05)。
(2)三叶人字草治疗IgA肾病的相关药效学研究：
高剂量(12.34 g／kg)、中剂量(6.17 g／k
结论：
三叶人字草能减少IgA肾病小鼠尿蛋白和血尿,降低IgA肾病小鼠血清中BUN、SCR含量,改善IgAN症状；能显著提高小鼠体内NO活力,能降低IgA肾病小鼠肾皮质中PDGF-B、PDGFR-B mRNA的表达,减少因PDGF-B及受体持续过度表达刺激系膜细胞增生及基质积累而导致肾脏疾病进行性发展,并能增强免疫,降压,止血,对胃肠功能有促进作用。三叶人字草对IgAN模型小鼠具有治疗作用,其表现出的肾脏保护效应,可能与抑制肾组织中PDGF-B、PDGFR-B mRNA的表达有重要作用,为其治疗IgA肾病的机制之一。
Objective:
Observe the effect of Kummerowia Striata from Denglao experience prescription on the expression of platelet-derived growth factor (PDGF-B) and its mRNA in IgA nephropathy model mouse. Determine the serum level of SCR、BUN、NO in IgA nephropathy model mice. Study the Kummerowia Striata's treatment on IgA nephropathy and the IgA nephropathy mechanism of pathogenesis. Besides, explore the pharmacodynamics of Kummerowia Striata treatment on IgAN and observe its effect on the immune system、blood pressure、blood function and gastrointestinal function etc. Provide experimental foundation for the clinical treatment of IgAN and promote the research level of traditional Chinese medicine in the nephritis research area. Advance the treatment of low toxicity and efficient of traditional Chinese medicine and provide a new method of treatment.
Method:
The extraction conditions were optimized by orthogonal test method. Investigated by orthogonal test method and level of different factors on the extraction rate, Preparation of this selection process with the Kummerowia Striata extract as the experimental drugs tested.
Using bovine serum albumin (BSA) and staphylococcal enterotoxin B (SEB) replicate IgA nephropathy mouse model. Observe the general situation of mice, serum SCR, BUN, NO level; determinate the expression of PDGF-B, PDGFR-B mRNA in IgA nephropathy mouse renal cortex by Semi-quantitative RT-PCR. Study the renal histopathology of Kummerowia Striata's treatment on IgA nephropathy model mice.
Effect of Kummerowia Striata on the immune system in normal mice was used by carbon clearance method. To study its effects on blood pressure by slide method. Study trefoil clotting and bleeding time and its platelet aggregation and other hemostatic factor by capillary method. Study the effects on gastrointestinal function to promote its treatment on IgA nephropathy pharmacodynamic by semi-solid method.
Results:
(1) pharmacological effects of Kummerowia Striata on IgA nephropathy model mice and its mechanism
As the Kummerowia Striata effect on IgA nephropathy model mice, the living conditions of mice significantly improved while proteinuria and the amout of hematuria reduced. The content serum of SCR, BUN decreased and the whole blood NO activity (P<0.05) increased in the high dose group(12.34 g/kg).The expression of mice renal cortex PDGF-B、PDGFR-B mRNA also reduced in the high dose group. Agarose gel eletrophoresis of products from PDGF-B、PDGFR-B mRNA gene RT-PCR of IgAN model mice showed that:193bp in each group were observed at the expression of PDGF-B mRNA,the other groups gradually darkened strip compared with model group. Semi-quantitative analysis showed that the expression of PDGF-B mRNA decreased 26.8% in KS high dose group with significant differences (P<0.05) compared with model group; 203bp in each group were observed at the expression of PDGFR-B mRNA, the other groups gradually darkened strip compared with model group. Semi-quantitative analysis showed that the expression of PDGF-B mRNA decreased 18.5% in KS high dose group with significant differences(P<0.05) compared with model group.
(2) Effect of Kummerowia Striata on IgA nephropathy model mice and its pharmacodynamics research
The mice peritoneal macrophage phagocytic clearance capacity and speed were significantly increased(P<0.01) in KS high dose group (12.34 g/kg) and medium dose group (6.17g/kg). The spleen of mice were significantly improved in KS high dose group and medium dose group. KS high dose could increase the normal mouse peritoneal macrophage phagocytosis (P<0.01). KS high、medium and low dose might have an effect on the rats blood pressure decreasing but have no significant difference compared with the former administration. KS high and medium dose could significantly shorten clotting time (slide method, capillary method) (P<0.01); KS high dose group mice have significantly effect of shorten the bleeding time (P<0.01) and increase platelet count (PLT) (P<0.01). But on the red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT) have no obvious indicators of the role of trend. KS high dose could significantly promote gastric emptying (P<0.01) and intestinal propulsiving (P<0.01).
(3)The best extraction of orthogonal filtering out KS:Water as extraction solvent,16 times the ratio of solid to liquid, boiling 0.5h, extract 3 times.
Conclusion:
KS could reduce proteinuria and the amout of hematuria in IgAN model mice, lower serum content of BUN, SCR and heal the symptoms of IgAN; significantly increase NO activity and reduce the renal cortex expression of PDGF-B and PDGFR-B mRNA in IgAN model mice;reduce the development of renal disease which is due to PDGF-B and its receptor stimulation sustained progressively over-expression of mesangial cell proliferation and matrix accumulation. KS also can enhance immunization, blood pressure, bleeding, promote gastrointestinal function. The protective effect of KS on IgAN model mice could be interrelated with the expression of PDGF-B and PDGFR-B mRNA that they might play an important role in IgAN and that may be one of the mechanisms of KS effect on IgAN.
引文
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