密蒙花方防治糖尿病视网膜病变TGF-β/Smad信号转导机制研究
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摘要
目的
从视网膜功能和细胞分子水平两个层次，系统观察中药复方密蒙花方对DM大鼠视网膜病变的作用和影响，并从信号转导机制方面深入研究其具体作用机制，为密蒙花方防治DR提供可靠的实验依据。
方法
选用链脲佐菌素(streptozocin，STZ)诱导的DM大鼠作为研究对象，以西药羟苯磺酸钙胶囊作为阳性对照药，将密蒙花方煎剂分为低、中、高三种不同浓度，在STZ性DM大鼠造模成功后即开始分别以上述药物干预治疗，灌胃4个月后，进行以下实验研究：
1．STZ性DM大鼠一般状况观察和检测：体毛、饮食、二便、体重、血糖等。
2．伊文思蓝法检测STZ性DM大鼠视网膜通透性的功能改变情况。
3．免疫组织化学法检测STZ性DM大鼠视网膜NF-κB、TGF-β_2蛋白表达情况。
4．RT-PCR法检测STZ性DM大鼠视网膜TGF-β_2 mRNA、Smad2 mRNA的表达变化情况。
5．Western blot法检测STZ性DM大鼠视网膜Smad2、P-Smad2蛋白表达变化情况。
结果
STZ性DM大鼠经药物干预4个月后，实验结果显示如下：
1．一般情况：大鼠经STZ诱导后，高血糖水平持久稳定，并表现出典型的“三多一少”症状。随着用药时间延长，密蒙花方中、低剂量组的一般DM症状较同期模型组明显改善，但对体重无改善(P＞0．05)；昊畅组与同期模型组比较能在一定程度上对抗STZ糖尿病大鼠体重的减轻(P＜0．05)。
2．血糖检测：DM模型组空腹血糖均显著高于同期正常对照组(P＜0．01)；随着用药时间延长，密蒙花方各剂量组空腹血糖较同期DM模型组降低，具有统计学意义(p＜0．05或P＜0．01)。其中，密蒙花方高剂量组血糖较低剂量组明显降低，具有统计学意义(p＜0．05)，而与中剂量比较无统计血意义(P＞0．05)。
3．视网膜通透性检测：密蒙花方中、高剂量组大鼠视网膜组织的EB含量与DM模型组比较都明显降低，差异有统计学意义(P＜0．01)，但二者之间比较无统计学意义(P＞0．05)；而密蒙花低剂量组、吴畅组与DM模型组比较无统计学意义(P＞0．05)。
4．免疫组化检测：①DM模型组视网膜NF-κB表达较正常对照组明显增多(P＜0．01)；密蒙花方各剂量组视网膜NF-κB表达较模型组、昊畅组均有不同程度的减少，差异有统计学意义(P＜0．01)，但密蒙花方低、中、高各剂量组之间比较均无统计学意义(P＞0．05)。②DM模型组视网膜TGF-β_2表达较正常对照组明显减少(P＜0．01)；密蒙花方低剂量组TGF-β_2表达量较模型组比较无统计学意义(p＞0．05)；密蒙花中剂量组TGF-β_2表达量较模型组、昊畅组比较具有统计学意义(p＜0．01)；密蒙花高剂量组TGF-β_2表达量较模型组、昊畅组比较具有统计学意义(p＜0．05)。密蒙花中、高剂量组TGF-β_2表达量比较无统计学意义(p＞0．05)。
5．RT-PCR结果显示：DM模型组TGF-β_2 mRNA、Smad2 mRNA表达较正常对照组表达明显减弱；吴畅组及密蒙花方各剂量组TGF-β_2 mRNA、Smad2 mRNA表达水平较正常对照组均不同程度表达减弱，但均较模型组表达水平增强。
6．Western blot检测结果显示：DM模型组Smad2、P-Smad2蛋白表达水平与正常对照组比较明显降低；与DM模型组比较，密蒙花方各剂量组及昊畅组Smad2、P-Smad2蛋白表达水平均不同程度升高。
结论
1．STZ诱导DM大鼠是一种相对简单易行、成模率高、可靠性强的动物模型制备方法。
2．密蒙花方能够改善STZ性DM大鼠的诸多糖尿病症状，但对体重无明显影响；吴畅在一定程度上具有对抗STZ性DM大鼠体重下降的作用。
3．密蒙花方对血糖升高具有一定的抑制作用，但这种作用是十分有限的。
4．密蒙花方能够在一定程度上延缓或降低大鼠视网膜毛细血管屏障的损害，减少视网膜血管的渗漏，改善视网膜的功能。
5．密蒙花方改善大鼠视网膜功能的作用可能与减少视网膜NF-κB及上调TGF-β_2表达有关。
6．密蒙花方改善大鼠视网膜功能的作用可能与上调TGF-β_2、Smad2、p-Smad2基因或蛋白表达，干预TGF-β/Smad信号转导通路有关。
Objective:
To investigate the effect and mechainsm of Mimeng Flower decoction on theretinal function and molecular change of diabetic rat,and further to study themechanism of signal transduction,in order to provide experimental basis forMimeng Flower decoction preventing and curing diabetic retinophty(DR).Methods:
We reproduced diabetic animal model by injecting of Streptozotozcin(STZ),and used calcium dobesilate as positive control medicine.Mimeng Flowerdecoction has been divided into three different concentration including lowdosage,median dosage and high dosage.Begaining with the blood glucose rising,wetreated the Chiese medicine groups with Mimeng Flower decoction and treated thecontrol group with calcium dobesilate.After 4 months,we made the followingexperimental studies:
1.To observe and test the general status of diabetic rats induced by STZ,suchas hairs,food and drink,urine and stool,weight,blood glucose and so on.
2.To evaluate the damage of blood-retina barrier(BRB) by using Evans blue totracing the leakage of retinal capillary barrier.
3.Through immunohistochemistry method to detect the protein expression of NF-κB、TGF-β_2 in retina of diabetic rats induced by STZ.
4.Through RT-PCR method to detect the expression of TGF-β_2 mRNA、Smad2 mRNA inretina of diabetic rats induced by STZ.
5.Through Western blot method to detect the expression of Smad2、P-Smad2 in retinaof diabetic rats induced by STZ.
Results
We draw the following conclusion after 4 months:1.General status:diabetic rats induced by STZ had lasting and stable hyperglycaemia,and displayed the obvious signs of“polydipsia,polyphagia,hyperdiuresis and loss of weight”.With the extension of treatment,mediandosage and low dosage of Mimeng Flower decoction could improve the typicaldiabetic symptoms compared with DM model group;the calcium dobesilate couldincrease the body weight of rats compared with homeochronous DM modelgroup(P＜0.05).
2.Blood glucose:Compared with homeochronous normal group,the blood glucose ofthe rats increaded remarkably in DM model group (P＜0.01);Compared with thehomeochronous DM model group,each dosage of Mimeng Flower decoction reduced theblood glucose of the rats (p＜0.05 or P＜0.01).
3.The results of the leakage of retinal capillary barrier show that the highdosage and median dosage of Mimeng Flower decoction reduced the leakage ofretinal capillary barrier of the rats,compared with the homeochronous DM modelgroup (p＜0.01).
4.The results of immunohistochemistry test show that the expression of NF-κB in retina increased in DM model group markedly compared with normal group(P＜0.01);Mimeng Flower decoction groups and the calcium dobesilate group coulddecrease the expression of NF-κB in retina compared with DM model grouprespectively(P＜0.01).The expression of TGF-β_2 in retina decreased in DM modelgroup markedly compared with normal group (P＜0.01);Mimeng Flower decoctiongroups could increase the expression of TGF-β_2 compared with DM model group andthe calcium dobesilate group (P＜0.01).
5.The results of RT-PCR test show that the expression of TGF-β_2 mRNA、Smad2 mRNAweakened in DM model group compared with normal group visibly;The expressionof TGF-β_2 mRNA、Smad2 mRNA in Mimeng Flower decoction groups and calciumdobesilate weakened in different extent compared with normal group,butall increased compared with DM model group.
6.The results of Western blot test show that the expression level of Smad2、P-Smad2 weakened in DM model group compared with normal group visibly;Theexpression level of Smad2、P-Smad2 in Mimeng Flower decoction groups and calcium dobesilate increased compared with DM model group in different extent.conclusion
1.STZ induced diabetic rat is a simple,high-ratio and reliable animol model.Mimeng Flower decoction could improve the typical diabetic symptoms comparedwith DM model group except the body weight,the calcium dobesilate could increasethe body weight of rats compared with homeochronous DM model group.
2.Mimeng Flower decoction could reduce the leakage of retinal capillary barrierof the DM rats.
3.The effect of improving the leakage of retinal capillary barrier of the DMrats may be contributed to the down regulation of NF-κB and the up regulationof TGF-β_2 in rat retina.
4.The up regulation of the mRNA expression and protein level of TGF-β_2、Smad2、p-Smad2 may play a significant role in improving the retinal function by MimengFlower decoction.
从视网膜功能和细胞分子水平两个层次，系统观察中药复方密蒙花方对DM大鼠视网膜病变的作用和影响，并从信号转导机制方面深入研究其具体作用机制，为密蒙花方防治DR提供可靠的实验依据。
方法
选用链脲佐菌素(streptozocin，STZ)诱导的DM大鼠作为研究对象，以西药羟苯磺酸钙胶囊作为阳性对照药，将密蒙花方煎剂分为低、中、高三种不同浓度，在STZ性DM大鼠造模成功后即开始分别以上述药物干预治疗，灌胃4个月后，进行以下实验研究：
1．STZ性DM大鼠一般状况观察和检测：体毛、饮食、二便、体重、血糖等。
2．伊文思蓝法检测STZ性DM大鼠视网膜通透性的功能改变情况。
3．免疫组织化学法检测STZ性DM大鼠视网膜NF-κB、TGF-β_2蛋白表达情况。
4．RT-PCR法检测STZ性DM大鼠视网膜TGF-β_2 mRNA、Smad2 mRNA的表达变化情况。
5．Western blot法检测STZ性DM大鼠视网膜Smad2、P-Smad2蛋白表达变化情况。
结果
STZ性DM大鼠经药物干预4个月后，实验结果显示如下：
1．一般情况：大鼠经STZ诱导后，高血糖水平持久稳定，并表现出典型的“三多一少”症状。随着用药时间延长，密蒙花方中、低剂量组的一般DM症状较同期模型组明显改善，但对体重无改善(P＞0．05)；昊畅组与同期模型组比较能在一定程度上对抗STZ糖尿病大鼠体重的减轻(P＜0．05)。
2．血糖检测：DM模型组空腹血糖均显著高于同期正常对照组(P＜0．01)；随着用药时间延长，密蒙花方各剂量组空腹血糖较同期DM模型组降低，具有统计学意义(p＜0．05或P＜0．01)。其中，密蒙花方高剂量组血糖较低剂量组明显降低，具有统计学意义(p＜0．05)，而与中剂量比较无统计血意义(P＞0．05)。
3．视网膜通透性检测：密蒙花方中、高剂量组大鼠视网膜组织的EB含量与DM模型组比较都明显降低，差异有统计学意义(P＜0．01)，但二者之间比较无统计学意义(P＞0．05)；而密蒙花低剂量组、吴畅组与DM模型组比较无统计学意义(P＞0．05)。
4．免疫组化检测：①DM模型组视网膜NF-κB表达较正常对照组明显增多(P＜0．01)；密蒙花方各剂量组视网膜NF-κB表达较模型组、昊畅组均有不同程度的减少，差异有统计学意义(P＜0．01)，但密蒙花方低、中、高各剂量组之间比较均无统计学意义(P＞0．05)。②DM模型组视网膜TGF-β_2表达较正常对照组明显减少(P＜0．01)；密蒙花方低剂量组TGF-β_2表达量较模型组比较无统计学意义(p＞0．05)；密蒙花中剂量组TGF-β_2表达量较模型组、昊畅组比较具有统计学意义(p＜0．01)；密蒙花高剂量组TGF-β_2表达量较模型组、昊畅组比较具有统计学意义(p＜0．05)。密蒙花中、高剂量组TGF-β_2表达量比较无统计学意义(p＞0．05)。
5．RT-PCR结果显示：DM模型组TGF-β_2 mRNA、Smad2 mRNA表达较正常对照组表达明显减弱；吴畅组及密蒙花方各剂量组TGF-β_2 mRNA、Smad2 mRNA表达水平较正常对照组均不同程度表达减弱，但均较模型组表达水平增强。
6．Western blot检测结果显示：DM模型组Smad2、P-Smad2蛋白表达水平与正常对照组比较明显降低；与DM模型组比较，密蒙花方各剂量组及昊畅组Smad2、P-Smad2蛋白表达水平均不同程度升高。
结论
1．STZ诱导DM大鼠是一种相对简单易行、成模率高、可靠性强的动物模型制备方法。
2．密蒙花方能够改善STZ性DM大鼠的诸多糖尿病症状，但对体重无明显影响；吴畅在一定程度上具有对抗STZ性DM大鼠体重下降的作用。
3．密蒙花方对血糖升高具有一定的抑制作用，但这种作用是十分有限的。
4．密蒙花方能够在一定程度上延缓或降低大鼠视网膜毛细血管屏障的损害，减少视网膜血管的渗漏，改善视网膜的功能。
5．密蒙花方改善大鼠视网膜功能的作用可能与减少视网膜NF-κB及上调TGF-β_2表达有关。
6．密蒙花方改善大鼠视网膜功能的作用可能与上调TGF-β_2、Smad2、p-Smad2基因或蛋白表达，干预TGF-β/Smad信号转导通路有关。
Objective:
To investigate the effect and mechainsm of Mimeng Flower decoction on theretinal function and molecular change of diabetic rat,and further to study themechanism of signal transduction,in order to provide experimental basis forMimeng Flower decoction preventing and curing diabetic retinophty(DR).Methods:
We reproduced diabetic animal model by injecting of Streptozotozcin(STZ),and used calcium dobesilate as positive control medicine.Mimeng Flowerdecoction has been divided into three different concentration including lowdosage,median dosage and high dosage.Begaining with the blood glucose rising,wetreated the Chiese medicine groups with Mimeng Flower decoction and treated thecontrol group with calcium dobesilate.After 4 months,we made the followingexperimental studies:
1.To observe and test the general status of diabetic rats induced by STZ,suchas hairs,food and drink,urine and stool,weight,blood glucose and so on.
2.To evaluate the damage of blood-retina barrier(BRB) by using Evans blue totracing the leakage of retinal capillary barrier.
3.Through immunohistochemistry method to detect the protein expression of NF-κB、TGF-β_2 in retina of diabetic rats induced by STZ.
4.Through RT-PCR method to detect the expression of TGF-β_2 mRNA、Smad2 mRNA inretina of diabetic rats induced by STZ.
5.Through Western blot method to detect the expression of Smad2、P-Smad2 in retinaof diabetic rats induced by STZ.
Results
We draw the following conclusion after 4 months:1.General status:diabetic rats induced by STZ had lasting and stable hyperglycaemia,and displayed the obvious signs of“polydipsia,polyphagia,hyperdiuresis and loss of weight”.With the extension of treatment,mediandosage and low dosage of Mimeng Flower decoction could improve the typicaldiabetic symptoms compared with DM model group;the calcium dobesilate couldincrease the body weight of rats compared with homeochronous DM modelgroup(P＜0.05).
2.Blood glucose:Compared with homeochronous normal group,the blood glucose ofthe rats increaded remarkably in DM model group (P＜0.01);Compared with thehomeochronous DM model group,each dosage of Mimeng Flower decoction reduced theblood glucose of the rats (p＜0.05 or P＜0.01).
3.The results of the leakage of retinal capillary barrier show that the highdosage and median dosage of Mimeng Flower decoction reduced the leakage ofretinal capillary barrier of the rats,compared with the homeochronous DM modelgroup (p＜0.01).
4.The results of immunohistochemistry test show that the expression of NF-κB in retina increased in DM model group markedly compared with normal group(P＜0.01);Mimeng Flower decoction groups and the calcium dobesilate group coulddecrease the expression of NF-κB in retina compared with DM model grouprespectively(P＜0.01).The expression of TGF-β_2 in retina decreased in DM modelgroup markedly compared with normal group (P＜0.01);Mimeng Flower decoctiongroups could increase the expression of TGF-β_2 compared with DM model group andthe calcium dobesilate group (P＜0.01).
5.The results of RT-PCR test show that the expression of TGF-β_2 mRNA、Smad2 mRNAweakened in DM model group compared with normal group visibly;The expressionof TGF-β_2 mRNA、Smad2 mRNA in Mimeng Flower decoction groups and calciumdobesilate weakened in different extent compared with normal group,butall increased compared with DM model group.
6.The results of Western blot test show that the expression level of Smad2、P-Smad2 weakened in DM model group compared with normal group visibly;Theexpression level of Smad2、P-Smad2 in Mimeng Flower decoction groups and calcium dobesilate increased compared with DM model group in different extent.conclusion
1.STZ induced diabetic rat is a simple,high-ratio and reliable animol model.Mimeng Flower decoction could improve the typical diabetic symptoms comparedwith DM model group except the body weight,the calcium dobesilate could increasethe body weight of rats compared with homeochronous DM model group.
2.Mimeng Flower decoction could reduce the leakage of retinal capillary barrierof the DM rats.
3.The effect of improving the leakage of retinal capillary barrier of the DMrats may be contributed to the down regulation of NF-κB and the up regulationof TGF-β_2 in rat retina.
4.The up regulation of the mRNA expression and protein level of TGF-β_2、Smad2、p-Smad2 may play a significant role in improving the retinal function by MimengFlower decoction.
引文
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