复方黄连胶囊对早期糖尿病肾病大鼠肾病变的防治作用研究
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摘要
目的:研究复方黄连胶囊对早期糖尿病肾病大鼠肾病变的防治作用。
方法:
1.药物制备和含量测定
1.1黄连中盐酸小檗碱提取:黄连粗品采用6倍量80%乙醇为溶剂,乙醇中加入0.25%硫酸,提取3次,每次90 min。
1.2葛根、枇杷叶、麦冬混合提取:葛根、枇杷叶、麦冬粗品采用8倍量60%乙醇为溶剂,提取3次,每次60min。
1.3采用HPLC测定CRCC中盐酸小檗碱的含量:选用C_(18)柱(250mm×4.6mm,5μm);检测波长263nm:流动相:0.033mol/LKH_2PO_(4-)乙腈(48:52):流速:1.0mL/min;柱温:25℃,进样量为20μL);
1.4采用HPLC测定CRCC中葛根素的含量:选用C_(18)柱(250mm×4.6mm,5μ);检测波长250nm:流动相:甲醇-水(26:74);流速:1.0mL/min:柱温:30℃,进样量为10μL):
2.DN大鼠模型建立、分组及给药:采用文献方法,选取健康Wistar雄性大鼠,体重为180～220g。禁食不禁水12h后,腹腔一次性无菌注射链脲佐菌素(STZ)60mg/kg,72h后眼眶取血测空腹血糖(FBG),选取血糖值≥11.1mmol/L,尿糖++～+++72只纳入实验。
符合条件动物随机均分为6组:模型组、CRCC高剂量组(4.36g/kg,相当于临床人用等效量4倍)、低剂量组(2.18 g/kg,相当于临床人用等效量2倍)、消渴丸组(0.80g/kg相当于临床人用等效量2倍)、盐酸小檗碱+葛根素组(盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg)、依那普利组(10mg/kg),另设正常对照组8只。
除模型组及正常对照组给予0.5%CMC-Na,其余各用药组均采用灌胃给药,一天一次,连续给药8w。
3.指标检测:生化分析:FBG、BUN、Scr:放免分析:Ins、C-P、TNF-α、α1-MG、β2-MG:光镜观察:肾小球HE染色;电镜观察:肾脏超微结构;免疫组化检测:肾皮质Bcl-2、Bax、VEGF蛋白表达:RT-PCR检测:肾皮质Bcl-2、Bax、VEGF mRNA表达:其他:体重、肾重、一般状态。
结果:模型组大鼠FBG、Scr、BUN、TNF-α、α1-MG、β2-MG水平显著升高,体重、Ins和C-P水平显著降低,光镜下可观察到模型组大鼠的肾小球细胞出现空泡样变,细胞外基质增多,毛细血管攀数量减少,肾小管肿胀。电镜下进一步观察到肾小球细胞基底膜增厚,足突融合,胞内线粒体出现退化性变,染色质出现边集现象。肾皮质Bcl-2蛋白和mRNA的表达降低,Bax、VEGF蛋白和mRNA的表达升高。上述结果提示,本实验成功制备了大鼠糖尿病肾病早期模型。与模型组比较,CRCC及其他各给药组能显著降低大鼠FBG、Scr、BUN、TNF-α、α1-MG、β2-MG水平,升高体重、Ins和C-P水平。光镜下观察,大鼠的肾小球细胞空泡样变程度减轻,细胞外基质堆积减少,开放的毛细血管数量增多,肾小管轻微肿胀。电镜下进一步观察到肾小球细胞基底膜增厚和足突融合程度减轻,线粒体轻度肿胀。肾皮质Bcl-2蛋白和mRNA的表达升高,Bax、VEGF蛋白和mRNA的表达降低。
结论:
1.CRCC(2.18、4.36g/kg),盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg,消渴丸(0.80g/kg)能够降低DN大鼠的FBG、升高Ins、C-P。
2.CRCC(2.18、4.36g/kg),盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg,消渴丸(0.80g/kg),依那普利(10mg/kg)能够降低DN大鼠Scr、BUN、α1-MG、β2-MG和肾重体重比值水平,改善肾功能。
3.病理观察提示CRCC(2.18、4.36g/kg),盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg,消渴丸(0.80g/kg),依那普利(10mg/kg)能够不同程度的促进DN大鼠肾小球结构逐步恢复和改善,保护肾脏超微结构。
4.RT-PCR结果提示CRCC(2.18、4.36g/kg),盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg,消渴丸(0.80g/kg),依那普利(10mg/kg)能够不同程度抑制肾皮质Bax、VEGF蛋白和mRNA表达,促进Bcl-2蛋白和mRNA表达。
以上实验结果综合表明,CRCC对早期DN具有防治作用,能够延缓DN进程。
OBJECTIVE:To research the preventive and therapeutic effect of compound Rhizoma coptidis capsule on early diabetic nephropathy rats.
METHODS:
1.Preparation and determination of CRCC:This experiment was designed on the basis of the preliminary work.We use established methods for preparation and determination of berberine hydrochloride and puerarin in compound Rhizoma Coptidis capsule。
2.Establishment,grouping and administration of DN rats:The early diabetic nephropathy rats were established by intraperitoneal injection in these rats with streptozotocin in a dose of 60mg/kg.Rats which FBG≥11.1mmol/L are used and divided into six groups randomly.CRCC(2.18,4.36g/kg),berberine hydrochloride 200mg/kg combinating puerarin 150mg/kg,XKW(0.8g/kg),Enalapril(10mg/kg) was administr -ated via gavage daily throughout the experiment.
3.Index detection:FBG、BUN、Scr are detected by biochemical analysis.The changes of Ins,C-P,TNF-α,α1-MG,β2-MG are reflected by RIA analysis.Light microscopy observe the glomerular HE staining and electron microscopy overview kidney ultrastructure.Immunohistochemistry express the renal cortex Bcl-2,Bax,VEGF protein content.RT-PCR test the expression of renal cortex Bcl-2,Bax,VEGF mRNA.
RESULTS:The results showed that serum levels of FBG,Scr,BUN,TNF-α,α1-MG,β2-MG markly increased,meanwhile the levels of bodyweight,INS and C-P markly decreased in model rats.The model rats' glomerular cells showed vacuolar changed, extracellular matrix increased and tubular swelled observed under light microscope. Glomerular basement membrane thickening and foot process integration were observed through the electron microscope.In conclusion,we successfully prepared the early diabetic nephropathy rat model.CRCC(2.18,4.36g/kg) not only obviously improve the glomerular cells showed vacuolar changed and reduce the accumulation of extracellular matrix,but also lessense the extent of glomerular basement membrane thickening.These results suggest that CRCC has positively preventive and therapeutic effect on DN rat.
Conclusion:To sum up,the streptozotocin induced early diabetic nephropathy rats model is more likely to reproduce the key features of early diabetic nephropathy in human.CRCC has positively preventive and therapeutic effect on experimental DN model.It can not only amendment the renal function,step down the blood glucose,heighten the insulinbut asoihas the ability to elevate the expression of Bc1-2 proteinum and mRNA, inhibit the Bax,VEGF proteinum and mRNA expression and protect the ultrastructural mitochondrial and renal lesion.
方法:
1.药物制备和含量测定
1.1黄连中盐酸小檗碱提取:黄连粗品采用6倍量80%乙醇为溶剂,乙醇中加入0.25%硫酸,提取3次,每次90 min。
1.2葛根、枇杷叶、麦冬混合提取:葛根、枇杷叶、麦冬粗品采用8倍量60%乙醇为溶剂,提取3次,每次60min。
1.3采用HPLC测定CRCC中盐酸小檗碱的含量:选用C_(18)柱(250mm×4.6mm,5μm);检测波长263nm:流动相:0.033mol/LKH_2PO_(4-)乙腈(48:52):流速:1.0mL/min;柱温:25℃,进样量为20μL);
1.4采用HPLC测定CRCC中葛根素的含量:选用C_(18)柱(250mm×4.6mm,5μ);检测波长250nm:流动相:甲醇-水(26:74);流速:1.0mL/min:柱温:30℃,进样量为10μL):
2.DN大鼠模型建立、分组及给药:采用文献方法,选取健康Wistar雄性大鼠,体重为180～220g。禁食不禁水12h后,腹腔一次性无菌注射链脲佐菌素(STZ)60mg/kg,72h后眼眶取血测空腹血糖(FBG),选取血糖值≥11.1mmol/L,尿糖++～+++72只纳入实验。
符合条件动物随机均分为6组:模型组、CRCC高剂量组(4.36g/kg,相当于临床人用等效量4倍)、低剂量组(2.18 g/kg,相当于临床人用等效量2倍)、消渴丸组(0.80g/kg相当于临床人用等效量2倍)、盐酸小檗碱+葛根素组(盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg)、依那普利组(10mg/kg),另设正常对照组8只。
除模型组及正常对照组给予0.5%CMC-Na,其余各用药组均采用灌胃给药,一天一次,连续给药8w。
3.指标检测:生化分析:FBG、BUN、Scr:放免分析:Ins、C-P、TNF-α、α1-MG、β2-MG:光镜观察:肾小球HE染色;电镜观察:肾脏超微结构;免疫组化检测:肾皮质Bcl-2、Bax、VEGF蛋白表达:RT-PCR检测:肾皮质Bcl-2、Bax、VEGF mRNA表达:其他:体重、肾重、一般状态。
结果:模型组大鼠FBG、Scr、BUN、TNF-α、α1-MG、β2-MG水平显著升高,体重、Ins和C-P水平显著降低,光镜下可观察到模型组大鼠的肾小球细胞出现空泡样变,细胞外基质增多,毛细血管攀数量减少,肾小管肿胀。电镜下进一步观察到肾小球细胞基底膜增厚,足突融合,胞内线粒体出现退化性变,染色质出现边集现象。肾皮质Bcl-2蛋白和mRNA的表达降低,Bax、VEGF蛋白和mRNA的表达升高。上述结果提示,本实验成功制备了大鼠糖尿病肾病早期模型。与模型组比较,CRCC及其他各给药组能显著降低大鼠FBG、Scr、BUN、TNF-α、α1-MG、β2-MG水平,升高体重、Ins和C-P水平。光镜下观察,大鼠的肾小球细胞空泡样变程度减轻,细胞外基质堆积减少,开放的毛细血管数量增多,肾小管轻微肿胀。电镜下进一步观察到肾小球细胞基底膜增厚和足突融合程度减轻,线粒体轻度肿胀。肾皮质Bcl-2蛋白和mRNA的表达升高,Bax、VEGF蛋白和mRNA的表达降低。
结论:
1.CRCC(2.18、4.36g/kg),盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg,消渴丸(0.80g/kg)能够降低DN大鼠的FBG、升高Ins、C-P。
2.CRCC(2.18、4.36g/kg),盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg,消渴丸(0.80g/kg),依那普利(10mg/kg)能够降低DN大鼠Scr、BUN、α1-MG、β2-MG和肾重体重比值水平,改善肾功能。
3.病理观察提示CRCC(2.18、4.36g/kg),盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg,消渴丸(0.80g/kg),依那普利(10mg/kg)能够不同程度的促进DN大鼠肾小球结构逐步恢复和改善,保护肾脏超微结构。
4.RT-PCR结果提示CRCC(2.18、4.36g/kg),盐酸小檗碱精制品200 mg/kg+葛根素150 mg/kg,消渴丸(0.80g/kg),依那普利(10mg/kg)能够不同程度抑制肾皮质Bax、VEGF蛋白和mRNA表达,促进Bcl-2蛋白和mRNA表达。
以上实验结果综合表明,CRCC对早期DN具有防治作用,能够延缓DN进程。
OBJECTIVE:To research the preventive and therapeutic effect of compound Rhizoma coptidis capsule on early diabetic nephropathy rats.
METHODS:
1.Preparation and determination of CRCC:This experiment was designed on the basis of the preliminary work.We use established methods for preparation and determination of berberine hydrochloride and puerarin in compound Rhizoma Coptidis capsule。
2.Establishment,grouping and administration of DN rats:The early diabetic nephropathy rats were established by intraperitoneal injection in these rats with streptozotocin in a dose of 60mg/kg.Rats which FBG≥11.1mmol/L are used and divided into six groups randomly.CRCC(2.18,4.36g/kg),berberine hydrochloride 200mg/kg combinating puerarin 150mg/kg,XKW(0.8g/kg),Enalapril(10mg/kg) was administr -ated via gavage daily throughout the experiment.
3.Index detection:FBG、BUN、Scr are detected by biochemical analysis.The changes of Ins,C-P,TNF-α,α1-MG,β2-MG are reflected by RIA analysis.Light microscopy observe the glomerular HE staining and electron microscopy overview kidney ultrastructure.Immunohistochemistry express the renal cortex Bcl-2,Bax,VEGF protein content.RT-PCR test the expression of renal cortex Bcl-2,Bax,VEGF mRNA.
RESULTS:The results showed that serum levels of FBG,Scr,BUN,TNF-α,α1-MG,β2-MG markly increased,meanwhile the levels of bodyweight,INS and C-P markly decreased in model rats.The model rats' glomerular cells showed vacuolar changed, extracellular matrix increased and tubular swelled observed under light microscope. Glomerular basement membrane thickening and foot process integration were observed through the electron microscope.In conclusion,we successfully prepared the early diabetic nephropathy rat model.CRCC(2.18,4.36g/kg) not only obviously improve the glomerular cells showed vacuolar changed and reduce the accumulation of extracellular matrix,but also lessense the extent of glomerular basement membrane thickening.These results suggest that CRCC has positively preventive and therapeutic effect on DN rat.
Conclusion:To sum up,the streptozotocin induced early diabetic nephropathy rats model is more likely to reproduce the key features of early diabetic nephropathy in human.CRCC has positively preventive and therapeutic effect on experimental DN model.It can not only amendment the renal function,step down the blood glucose,heighten the insulinbut asoihas the ability to elevate the expression of Bc1-2 proteinum and mRNA, inhibit the Bax,VEGF proteinum and mRNA expression and protect the ultrastructural mitochondrial and renal lesion.
引文
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