夏枯草提取物对尿草酸钙结石形成及肾结石大鼠Osteopontin表达的影响
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摘要
肾结石是一种临床常见疾病,结石的主要成份为草酸钙。现代医学认为尿液中草酸钙结晶的成核、生长、聚集及对肾小管上皮细胞的黏附在肾结石的形成过程中起重要作用。研究发现尿液中含有多种抑制草酸钙结晶形成的大分子物质,骨桥蛋白(osteopontin,OPN)是其中一种代表性的蛋白质。夏枯草是一种有效治疗尿石症的传统中草药,但其具体作用机制尚不清楚,在本研究中我们探讨了夏枯草不同提取物对草酸钙结晶形成的体外和体内影响,并研究夏枯草提取物对大鼠肾组织OPN表达的影响。
目的:探讨中药夏枯草不同提取物体外抑制尿草酸钙结石形成的效应以及体内对大鼠尿草酸钙结石形成的影响;并研究夏枯草提取物对大鼠OPN表达的影响,初步探讨夏枯草抑制尿草酸钙结石形成的机制。
方法:体外实验采用种晶技术检测不同浓度(10和20mg·ml~(-1))夏枯草水溶性提取物(C_1、C_2组)、50%甲醇提取物(D_1、D_2组)和100%甲醇提取物(E_1、E_2组)对体外一水草酸钙晶体生长的抑制指数。40只Wistar大鼠随机分为5组(每组8只),采用乙二醇和氯化铵诱导的大鼠肾草酸钙结石模型进行试验研究。对照组(A组)、成石组(B组)、夏枯草水溶性提取物组(C组)、50%甲醇提取物组(D组)、100%甲醇提取物组(E组),分别通过灌胃方式予一定剂量的夏枯草不同提取物。饲养4周后,检测各组大鼠血BUN、Cr,24h尿草酸(Ox)、Ca~(2+)分泌量和肾组织Ca~(2+)含量。镜下观察肾组织切片中草酸钙结晶及肾小管扩张情况,检测各组大鼠肾组织病理学改变和草酸钙结晶沉积情况,并采用原位杂交和逆转录聚合酶链反应(RT-PCR)分别观察各组大鼠肾组织中OPN mRNA表达,免疫组织化学和蛋白质印迹(Western blotting)检测各组大鼠肾组织OPN蛋白的表达水平。
结果:体外实验显示夏枯草不同提取液体外对草酸钙结晶的抑制影响随药物浓度增加,其对草酸钙结晶生长的抑制作用也增强,且组间差异具有统计学意义(P＜0.05)。在相同药物浓度条件下,夏枯草水提取物和50%甲醇提取物对草酸钙结晶生长的抑制作用较强,而100%甲醇提取组的抑制作用相对较低。
动物实验显示A组大鼠血BUN、Cr明显低于其他各组,差别有显著性意义(P＜0.05),各组间血Ca~(2+)、P浓度则无明显差异;24h尿Ox、Ca~(2+)分泌量各夏枯草实验组与B组无明显差异(P＞0.05);C、D组肾组织Ca~(2+)含量明显低于B组,差别有显著性意义(P＜0.05)。A组肾小管正常,B组肾小管腔可见大量成片草酸钙结晶存在,管腔明显扩张;C组肾小管腔可见散在草酸钙结晶存在,少数呈片状,管腔轻度扩张;D组肾小管腔仅见少许散在草酸钙结晶存在,管腔未见明显扩张;E组基本同B组。
原位杂交显示OPN mRNA主要在肾近曲小管和远曲小管,亨利氏袢,和集合导管(主要髓袢升支)明显表达,而在皮质肾小球中则未能检测到OPN mRNA的表达。A组大鼠肾组织中仅肾脏髓质小部分亨利氏袢中检测到OPN mRNA的表达,与此相反在B组和D理组大鼠肾组织中则明显检测到OPN mRNA的表达,只是B组中OPNmRNA的表达强度相对弱于成石对照组。RT-PCR检测结果与原位杂交一致。免疫组化显示OPN蛋白在所有3组中均有表达,在肾近曲小管和远曲小管,亨利氏袢,和集合导管(主要髓袢升支)明显表达OPN蛋白,而在皮质肾小球中则未能检测到OPN蛋白的表达。在正常对照组中仅能检测到OPN蛋白的微弱表达。但是在B组大鼠肾组织中OPN表达则明显增强。D组大鼠肾组织中OPN表达强度低于成石对照组。Western blotting检测显示A组轻度表达OPN,而B组则显著表达,D组表达强度低于结石组。
结论:夏枯草水提取物和50%甲醇提取物体外能够有效抑制尿草酸钙结石形成。体内实验表明夏枯草50%甲醇提取物能有效防止大鼠体内尿草酸钙结石形成。夏枯草50%甲醇提取物能有效抑制大鼠肾组织OPN的表达,减少肾组织草酸钙结晶的沉积,从而能有效抑制大鼠肾草酸钙结石的形成。
Calcium nephrolithiasis is the most common form of renal stone disease,with calcium oxalate(CaOx)being the predominant constituent of renal stones.Modern medicine consider that CaOx crystal nucleation, growth,aggregation,and adhesion to renal cells plays a important role during renal stones formation and retention.Current in vitro evidence implicates osteopontin(OPN)as one of several macromolecular inhibitors of urinary crystallization with potentially important actions at several stages of CaOx crystal formation and retention.Prunella vulgaris L is a traditional chinese herbal used to treat various diseases for hundreds of years.Chinese herbal had been also used for the cure and the prevention of urinary calculi for many years,but the effect and the mechanism of this use of chinese herbal medicine are unclear.In the study we examined the inhibitory effect of the Chinese herbal medicine Prunella vulgaris L on the formation of CaOx crystral in vitro and CaOx renal stones induced by ethylene glycol(EG)and ammonium chloride in rats in vivo.We also investigated the e.ect of Prunella vulgaris L on osteopontin(OPN)expression.
Objective:To study the inhibitory effect of Prunella vulgaris on urinary oxalate calcium(CaOx)stone formation in vitro and to investigate the effects the extracts from Prunella vulgaris on urinary calcium oxalate stone formation and osteopont in(OPN)expression in urolithiasis model rats,and to explore the mechanism of Prunella vulgaris on the prevention of the formation from urinary calculi.
Methods:The aqueous extract from Prunella vulgaris(Aq-P.V) (group C_1and C_2),50%methanolic extract from Prunella vulgaris (Me-P.V)(group D_1and D_2),and 100%Me-P.V(group E_1and E_2)were prepared.The effects of several extracts whose concentration were at 10 and 20 mg·ml~(-1),respectively,on inhibiting CaOx crystal growth (inhibiting index,I.I)were determined by a seeded crystallization technique in vitro.
Forty adult male wistar strain rats were randomly divided into five groups(N=8 each),group normal control(A),group stone formation(B), Group TAq-ect(C),group 50%TMe-ext(D),group 100%TMe-ext(E). The model rats with renal calcium oxalate stone formation were induced by intragastric administration ethylene glycol and ammonium chloride. After 4 weeks,the concentration of serum BUN,Cr,Ca~(2+),P,the 24h urinary excretion Ca~(2+),Oxalate(Ox),Mg~(2+)and the content of renal tissue Ca~(2+),Mg~(2+)were detected.All kidneys were removed and examined microscopically for possible CaOx crystals deposit and tubular dilatation in the kidney.
The expression of OPN mRNA in the rats kidney were detected by in situ hybridization and RT-polymerase chain reaction(RT-PCR) techniques.Immunohistochemistry and western blotting methods were used to assessed the protein expression of OPN in rat renal tissue of every groups.
Results:The in vitro experiment showed that I.I in group C_1,C_2, D_1,D_2,E_1,E_2 was 50.8±5.6%,79.6±4.5%,45.6±6.5%,77.2±5.9%, 30.6±4.6%and 68.5±5.8%,respectively.The Aq-P.V had the strongest inhibition with I.I of 79.6±4.5%in high concentration,and I.I of group C and D were more than group E,but there was no significant difference in all groups in low concentration.
The serum concentration of Ca~(2+)and P showed no significant difference in all rats.24h urinary excretion Ca~(2+),Ox and Mg~(2+)showed no significant difference compared each group of administered extract from Prunella vulgaris with group B.The content of renal tissue Ca~(2+)was significantly lower in group A,C,D compared with group B(P＜0.05), the content of renal Mg~(2+)showed no significant difference in all groups. A large number of CaOx crystals and significant tubular dilatation were observed in group B.CaOx crystals and tubular dilatation in group C were significantly difference compared with group B,only a few CaOx crystals and few tubular dilatation were observed in group D,group E were analogous with group B.
von Kossa's method clearly demonstrated the presence of calcium oxalate deposits in the kidney of the stone group and Prunella vulgaris group.No deposits were detected in the control rats.Quantitative analysis of the density of calcium deposits showed a significantly lower density of deposits in the Prunella vulgaris group compared to the stone group.In situ hybridization demonstrated the expression of OPN mRNA in both the distal and proximal convoluted tubules,the loop of Henle,and collecting ducts(mainly the medullary thick ascending limb of the loop of Henle), the glomeruli were negative.In kidney from the normal control group, OPN mRNA was detected in a small proportion of the loops of Henle in the renal medulla.The kidney from the stone and 50%TMe-ext group showed overexpression of OPN mRNA;the increase in the 50%TMe-ext group was relatively weak compared to the stone group.The expression level of OPN mRNA using RT-PCR was correspond to In situ hybridization.The OPN protein expression was observed in the kidney of all three groups rats.The OPN protein were found in both the distal and proximal convoluted tubules,the loop of Henle,and collecting ducts(mainly the medullary thick ascending limb of the loop of Henle), whereas no staining for the protein was detected in glomeruli in the renal cortex.In rats of normal control group,only a weak OPN staining was noted.The expression was enhanced in the stone group.50%TMe-ext-treated rats tended to have lower OPN protein expression than the stone group.It was similar to Immunohistochemistry staining that the expression of OPN protein detected by western blotting.
Conclusion:The extract from Prunella vulgaris can significantly inhibited urinary CaOx stone formation in vitro,the inhibitory effect of Aq-P.V and 50%Me-P.V were better than 100%Me-P.V.Animal experimental showed that 50%Me-P.V can more significantly decrease the content of the renal tissue Ca~(2+)and tubular dilatation than Aq-P.V did in the in vivo experiment.The extracts from 50%Me-P.V Prunella vulgaris can inhibit the OPN expression and deposit of calcium oxalate in renal tissue so as to block the formation of renal calcium oxalate stone in rats potently.
目的:探讨中药夏枯草不同提取物体外抑制尿草酸钙结石形成的效应以及体内对大鼠尿草酸钙结石形成的影响;并研究夏枯草提取物对大鼠OPN表达的影响,初步探讨夏枯草抑制尿草酸钙结石形成的机制。
方法:体外实验采用种晶技术检测不同浓度(10和20mg·ml~(-1))夏枯草水溶性提取物(C_1、C_2组)、50%甲醇提取物(D_1、D_2组)和100%甲醇提取物(E_1、E_2组)对体外一水草酸钙晶体生长的抑制指数。40只Wistar大鼠随机分为5组(每组8只),采用乙二醇和氯化铵诱导的大鼠肾草酸钙结石模型进行试验研究。对照组(A组)、成石组(B组)、夏枯草水溶性提取物组(C组)、50%甲醇提取物组(D组)、100%甲醇提取物组(E组),分别通过灌胃方式予一定剂量的夏枯草不同提取物。饲养4周后,检测各组大鼠血BUN、Cr,24h尿草酸(Ox)、Ca~(2+)分泌量和肾组织Ca~(2+)含量。镜下观察肾组织切片中草酸钙结晶及肾小管扩张情况,检测各组大鼠肾组织病理学改变和草酸钙结晶沉积情况,并采用原位杂交和逆转录聚合酶链反应(RT-PCR)分别观察各组大鼠肾组织中OPN mRNA表达,免疫组织化学和蛋白质印迹(Western blotting)检测各组大鼠肾组织OPN蛋白的表达水平。
结果:体外实验显示夏枯草不同提取液体外对草酸钙结晶的抑制影响随药物浓度增加,其对草酸钙结晶生长的抑制作用也增强,且组间差异具有统计学意义(P＜0.05)。在相同药物浓度条件下,夏枯草水提取物和50%甲醇提取物对草酸钙结晶生长的抑制作用较强,而100%甲醇提取组的抑制作用相对较低。
动物实验显示A组大鼠血BUN、Cr明显低于其他各组,差别有显著性意义(P＜0.05),各组间血Ca~(2+)、P浓度则无明显差异;24h尿Ox、Ca~(2+)分泌量各夏枯草实验组与B组无明显差异(P＞0.05);C、D组肾组织Ca~(2+)含量明显低于B组,差别有显著性意义(P＜0.05)。A组肾小管正常,B组肾小管腔可见大量成片草酸钙结晶存在,管腔明显扩张;C组肾小管腔可见散在草酸钙结晶存在,少数呈片状,管腔轻度扩张;D组肾小管腔仅见少许散在草酸钙结晶存在,管腔未见明显扩张;E组基本同B组。
原位杂交显示OPN mRNA主要在肾近曲小管和远曲小管,亨利氏袢,和集合导管(主要髓袢升支)明显表达,而在皮质肾小球中则未能检测到OPN mRNA的表达。A组大鼠肾组织中仅肾脏髓质小部分亨利氏袢中检测到OPN mRNA的表达,与此相反在B组和D理组大鼠肾组织中则明显检测到OPN mRNA的表达,只是B组中OPNmRNA的表达强度相对弱于成石对照组。RT-PCR检测结果与原位杂交一致。免疫组化显示OPN蛋白在所有3组中均有表达,在肾近曲小管和远曲小管,亨利氏袢,和集合导管(主要髓袢升支)明显表达OPN蛋白,而在皮质肾小球中则未能检测到OPN蛋白的表达。在正常对照组中仅能检测到OPN蛋白的微弱表达。但是在B组大鼠肾组织中OPN表达则明显增强。D组大鼠肾组织中OPN表达强度低于成石对照组。Western blotting检测显示A组轻度表达OPN,而B组则显著表达,D组表达强度低于结石组。
结论:夏枯草水提取物和50%甲醇提取物体外能够有效抑制尿草酸钙结石形成。体内实验表明夏枯草50%甲醇提取物能有效防止大鼠体内尿草酸钙结石形成。夏枯草50%甲醇提取物能有效抑制大鼠肾组织OPN的表达,减少肾组织草酸钙结晶的沉积,从而能有效抑制大鼠肾草酸钙结石的形成。
Calcium nephrolithiasis is the most common form of renal stone disease,with calcium oxalate(CaOx)being the predominant constituent of renal stones.Modern medicine consider that CaOx crystal nucleation, growth,aggregation,and adhesion to renal cells plays a important role during renal stones formation and retention.Current in vitro evidence implicates osteopontin(OPN)as one of several macromolecular inhibitors of urinary crystallization with potentially important actions at several stages of CaOx crystal formation and retention.Prunella vulgaris L is a traditional chinese herbal used to treat various diseases for hundreds of years.Chinese herbal had been also used for the cure and the prevention of urinary calculi for many years,but the effect and the mechanism of this use of chinese herbal medicine are unclear.In the study we examined the inhibitory effect of the Chinese herbal medicine Prunella vulgaris L on the formation of CaOx crystral in vitro and CaOx renal stones induced by ethylene glycol(EG)and ammonium chloride in rats in vivo.We also investigated the e.ect of Prunella vulgaris L on osteopontin(OPN)expression.
Objective:To study the inhibitory effect of Prunella vulgaris on urinary oxalate calcium(CaOx)stone formation in vitro and to investigate the effects the extracts from Prunella vulgaris on urinary calcium oxalate stone formation and osteopont in(OPN)expression in urolithiasis model rats,and to explore the mechanism of Prunella vulgaris on the prevention of the formation from urinary calculi.
Methods:The aqueous extract from Prunella vulgaris(Aq-P.V) (group C_1and C_2),50%methanolic extract from Prunella vulgaris (Me-P.V)(group D_1and D_2),and 100%Me-P.V(group E_1and E_2)were prepared.The effects of several extracts whose concentration were at 10 and 20 mg·ml~(-1),respectively,on inhibiting CaOx crystal growth (inhibiting index,I.I)were determined by a seeded crystallization technique in vitro.
Forty adult male wistar strain rats were randomly divided into five groups(N=8 each),group normal control(A),group stone formation(B), Group TAq-ect(C),group 50%TMe-ext(D),group 100%TMe-ext(E). The model rats with renal calcium oxalate stone formation were induced by intragastric administration ethylene glycol and ammonium chloride. After 4 weeks,the concentration of serum BUN,Cr,Ca~(2+),P,the 24h urinary excretion Ca~(2+),Oxalate(Ox),Mg~(2+)and the content of renal tissue Ca~(2+),Mg~(2+)were detected.All kidneys were removed and examined microscopically for possible CaOx crystals deposit and tubular dilatation in the kidney.
The expression of OPN mRNA in the rats kidney were detected by in situ hybridization and RT-polymerase chain reaction(RT-PCR) techniques.Immunohistochemistry and western blotting methods were used to assessed the protein expression of OPN in rat renal tissue of every groups.
Results:The in vitro experiment showed that I.I in group C_1,C_2, D_1,D_2,E_1,E_2 was 50.8±5.6%,79.6±4.5%,45.6±6.5%,77.2±5.9%, 30.6±4.6%and 68.5±5.8%,respectively.The Aq-P.V had the strongest inhibition with I.I of 79.6±4.5%in high concentration,and I.I of group C and D were more than group E,but there was no significant difference in all groups in low concentration.
The serum concentration of Ca~(2+)and P showed no significant difference in all rats.24h urinary excretion Ca~(2+),Ox and Mg~(2+)showed no significant difference compared each group of administered extract from Prunella vulgaris with group B.The content of renal tissue Ca~(2+)was significantly lower in group A,C,D compared with group B(P＜0.05), the content of renal Mg~(2+)showed no significant difference in all groups. A large number of CaOx crystals and significant tubular dilatation were observed in group B.CaOx crystals and tubular dilatation in group C were significantly difference compared with group B,only a few CaOx crystals and few tubular dilatation were observed in group D,group E were analogous with group B.
von Kossa's method clearly demonstrated the presence of calcium oxalate deposits in the kidney of the stone group and Prunella vulgaris group.No deposits were detected in the control rats.Quantitative analysis of the density of calcium deposits showed a significantly lower density of deposits in the Prunella vulgaris group compared to the stone group.In situ hybridization demonstrated the expression of OPN mRNA in both the distal and proximal convoluted tubules,the loop of Henle,and collecting ducts(mainly the medullary thick ascending limb of the loop of Henle), the glomeruli were negative.In kidney from the normal control group, OPN mRNA was detected in a small proportion of the loops of Henle in the renal medulla.The kidney from the stone and 50%TMe-ext group showed overexpression of OPN mRNA;the increase in the 50%TMe-ext group was relatively weak compared to the stone group.The expression level of OPN mRNA using RT-PCR was correspond to In situ hybridization.The OPN protein expression was observed in the kidney of all three groups rats.The OPN protein were found in both the distal and proximal convoluted tubules,the loop of Henle,and collecting ducts(mainly the medullary thick ascending limb of the loop of Henle), whereas no staining for the protein was detected in glomeruli in the renal cortex.In rats of normal control group,only a weak OPN staining was noted.The expression was enhanced in the stone group.50%TMe-ext-treated rats tended to have lower OPN protein expression than the stone group.It was similar to Immunohistochemistry staining that the expression of OPN protein detected by western blotting.
Conclusion:The extract from Prunella vulgaris can significantly inhibited urinary CaOx stone formation in vitro,the inhibitory effect of Aq-P.V and 50%Me-P.V were better than 100%Me-P.V.Animal experimental showed that 50%Me-P.V can more significantly decrease the content of the renal tissue Ca~(2+)and tubular dilatation than Aq-P.V did in the in vivo experiment.The extracts from 50%Me-P.V Prunella vulgaris can inhibit the OPN expression and deposit of calcium oxalate in renal tissue so as to block the formation of renal calcium oxalate stone in rats potently.
引文
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