中药联合碳酸锂防治大鼠草酸钙肾结石药理作用及其机制研究
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摘要
目的结石形成是一个漫长、多因素共同作用过程,包括结晶的成核、生长、聚集和滞留。由于受地理、气候、种族和饮食习惯等多种因素影响,全球尿系结石患病率为1%-20%,平均约为10%。随着微创外科技术的广泛临床应用,大多数结石患者得到充分而有效的治疗,但其术后高复发率和并发症尚未有效解决。虽然柠檬酸盐和噻嗪类利尿剂具有一定的抑制结石形成作用,但其明显的副作用限制临床应用。因此寻求一种安全有效、价格低廉、适宜长期服用的预防结石药物,成为人们一直试图解决的问题。近年来,紫杉醇、银杏叶、喜树碱和青蒿素等一批以植物药为基础研制开发的新药应用于临床,使抗结石植物药物的开发研究方兴未艾,成为人们研究热点。在祖国医学中,金钱草和石韦是具有缓解疼痛、减少血尿及促进排石等功效的经典中草药。另有研究表明,锂离子通过二羧酸转运蛋白抑制肾脏对尿柠檬酸重吸收作用。在我们前期动物和临床实验研究中,碳酸锂显示出良好的内源性调节尿柠檬酸水平效果。由于结石形成是多因素共同作用结果,我们拟将中药的多作用靶点和碳酸锂单作用靶点的特点结合起来,联合应用以提高对尿柠檬酸浓度调节作用,增强预防大鼠肾结石形成,筛选最佳组配,并探讨其可能作用机制。
方法80只成年雄性Wistar大鼠随机分入八组(正常、对照、低中高剂量金钱草和石韦组)。经一周适应环境,5%草酸铵饲料喂养实验组大鼠一周后,以不同浓度水煎剂灌胃(1ml／次,2次／天)。第21天置入代谢笼留取尿液／24h标本后,10%水合氯醛腹腔注射麻醉大鼠后,经下腹部血管采集血液,摘取肾脏标本。检测血、尿标本各项理化指标,观察尿结晶、肾结晶沉积和病理改变,并测定肾和尿液中骨桥蛋白表达及肾组织氧化应激指标。另取,90只成年雄性Wistar大鼠随机分入九组(正常、对照、低中高剂量金钱草组、碳酸锂组和低中高剂量金钱草联合碳酸锂组)。干预方法同上留取血、尿和肾组织标本。检测血、尿标本各项理化指标,观察尿结晶、肾结晶沉积和病理改变。
结果结石模型大鼠尿草酸水平升高3倍,尿钙水平下降5倍。中高剂量金钱草组尿量／24h、尿柠檬酸浓度明显升高,尿钙和草酸浓度下降(P<0.05)。在高剂量石韦组血钙和尿钙水平均有不同程度下降。正常大鼠肾组织几乎没有结晶沉积和病理改变,尿中亦无草酸钙结晶,亨利氏袢和肾小管上皮细胞表面OPN蛋白呈弱表达。结石大鼠肾组织多部位存在结晶沉积(以皮髓质交界处为主),肾小管显著扩张并结晶簇存在,并伴有大量炎细胞浸润；尿液中存在大量长柱形的一水草酸钙和菱形的二水草酸钙结晶。在中高剂量金钱草组,相对于结石模型大鼠而言,肾组织结晶沉积评分明显降低,而两组之间没有统计学差异；病理仅表现出小管轻度扩张和炎症浸润,OPN蛋白表达降低。尿晶体数量明显减少,分别为4.63±2.33、5.67±1.73和14.6±3.72(P<0.001)。高剂量石韦组呈现相似变化,但炎细胞浸润明显减少。结石大鼠NO和MDA水平迅速升高,SOD活性则被抑制(P<0.05)。尽管在高剂量金钱草和中剂量石韦组NO水平下降,SOD活性得到恢复,但MDA水平没有发生改变。在中剂量金钱草和高剂量石韦组上述指标均恢复接近正常值。仅在碳酸锂及联合治疗组检测到血清锂水平,且呈逐渐增高趋势,高剂量金钱草联合治疗组为1.38±0.41mmol／L。在碳酸锂和中高剂量联合治疗组,尿pH值、柠檬酸、钙水平明显升高。中高剂量联合治疗组的尿柠檬酸水平较中高剂量金钱草组增高53%,较碳酸锂组增高24.3%,尿量／24h增加1倍,尿草酸水平降低1倍；光镜下偶见结晶体的沉积,结晶评分明显降低(P<0.05)。除低剂量金钱草组外,各实验组病理评分均显著降低,其中以中剂量联合治疗组评分最低为0.78±0.67。中高剂量联合治疗组尿中晶体分别减少至1.11±1.05和1.38±1.06。
结论金钱草作为传统治疗泌尿系结石的植物药,具有利尿、增加尿柠檬酸、降低尿草酸、升高尿钙水平和抗氧化作用,在中高剂量显示出明显的抑制结石形成作用。由于两者效果相似,故认为中剂量效果更佳。高剂量石韦有抗炎,抗氧化作用及相对较弱抑制结石形成作用。中剂量金钱草联合碳酸锂组对尿柠檬酸浓度表现出显著的协同调节作用,使尿柠檬酸水平较正常大鼠升高415%,较中高剂量金钱草升高53%,较单剂量碳酸锂升高24.3%。此外,尿量增加、尿pH值升高、尿草酸水平降低均有利于抑制结-石形成。
Objective The pathogenesis of CaOx stone is a gradually multifaceted process including crystal nucleation, growth, aggregation and retention. The prevalence of urinary stone ranged from 1% to 20% (average 10%) in the world wide due to various factors, such as geography, climate, race and diet. Minimally invasive techniques were widely applied in clinic so that many patients with urolithiasis gained an effective management. However, both the high recurrence rate and complications are so far a serious problem and remains to be decreased. Even though thiazide diuretics and alkali-citrate have been proven to be effective in reducing calciuria and stone recurrence, undesirable side-effects limit their use in long-term medical treatment. Therefore, finding an effective alternative with few adverse-effects is becoming a new tendency. Currently, there has been a sustained concern on medicinal plants to prevent stone recurrence owing to their efficiency, economy, and safety. Recently, the new drugs, which based on medicinal plants, such as Paclitaxel, Ginkgo biloba, Camptothecin, Artemisinin, and so on, are used in clinic and cause a resurgence of interest in medicinal plants. In traditional chinese medicine (TCM), Desmodium styracifolium (Ds) and Pyrrosiae petiolosa (Pp) have been widely used to release renal colic, hematuria, and remove stones. Additionally, our previous studies has shown that Lithium carbonate can significantly modulate endogenous citrate level according to lithium inhibit sodium dicarboxylate co-transporters (NaDC), which can transport citrate from tubular into cells. Due to calculus is the result of multifactor, we propose to increase citrate regulation through the combination the multi-target effect of chinese herbs and single target effect of LiC, enhance the prophylaxis of CaOx stones, select the optimal formula and clear its possible mechanism.
Methods 80 adult male Wistar rats were randomly divided into eight groups (Normal, Control, LDs, MDs, HDs, LPp, MPp, and HPp). After one week of acclimatization, the rats were fed by containing 5% ammonium oxalate (AmOx) feed for one week, and then given the different does aqueous extracts(lml/time, Bid). On the 21st day of experimental period, the rats were placed in separate metabolic cages to collect 24-h urine, and then were anesthetized intraperitoneally with 10% Chloral Hydrate. Blood was obtained via inferior vena cava and then immediately excised kidneys. Samples were used to various determinations involving physical and biochemtry examination, pathological examination, crystalluria and CaOx deposition evaluation, OPN immunohistochemical staining, and oxidative stress studies.And then,90 adult male Wistar rats were randomly divided into eight groups (Normal, Placebo, LDs, MDs, HDs, LiC, LDs+LiC, MDs+LiC, HDs+LiC). The same interventions were 'used to treat the rats with the different doses aqueous extracts. The blood,24-h urine and kidneys were collected and used to various determinations involving physical and biochemtry examination, crystalluria, CaOx deposition evaluation and pathological examination.
Results There were 3-fold increase in urinary Ox level and 5-fold decrease in Ca excretion in the urolithic rats.24h urinary volume, Ca, and Cit level were increased with varying degrees in the MDs and HDs group, and Ox concentration decreased (P<0.05). Not only was serum Ca distinctly decreased, but. also urinary Ca reduced in HPp group. In the normal group, no CaOx deposition was detected without pathological injury and CaOx crystalluria. OPN protein expression was weak and observed in the loop of Henle and papillary surface epithelium of few cells. Urolithic rats were observed more crystals dispositions in all parts of the kidney, especially in cortico-medullary junction, severe dilation of tubules and massive inflammatory infiltration, and more columnar whewellite and pyramidal weddellite crystals existed in urine. Scoring of crystal deposition showed a significantly lower number of deposits in MDs and HDs groups, but no statistic difference was concluded between two groups. In MDs and HDs groups, scoring of crystal deposition showed a significantly lower number of deposits, but no statistic difference was concluded between two groups. Pathological alteration reduced and has slight dilation of tubules and inflammatory infiltration. Simultaneously, OPN showed significantly lower expression than those in the urolithiatic rats. The mean number of crystals was distinctly reduced (MDs, HDs vs. Control:4.63±2.33,5.67±1.73 vs.14.6±3.72, P<0.001). Similar results as well as slight inflammatory infiltration were found in high dose Pp group. NO and MDA level rose rapidly, but SOD activities were inhibited in the control group (P<0.05). Although lower NO and higher SOD level were significantly observed in HDs and MPp groups, there was no Statistical significance in MDA. However, in MDs and HPp groups above three indicators restored to near normal levels. Serum Li was only detected in Lic and combination therapy groups, and showed a gradually increasing trend. Moreover, serum Li level was 1.38±0.41mmol/L in high dose combination therapy group. Urinary pH value, citrate, and calcium concentration significantly increased. Of these, urinary citrate level increased 53% and 24.3% compared with Ds and LiC groups, respectively. There was 1-fold increase in urinary volume and 1-fold decrease in Ox excretion. Crystal depositions were few, and its score showed a significantly lower number of deposits in MDs+LiC and HDs+LiC groups(P<0.05). The score of experiment groups except for LDs group demonstrated distinct reduction, and the score of MDs+LiC is lowest (0.78±0.67). Urinary crystals reduced to 1.11±1.05 and 1.38±1.06.
Conclusions DS as a traditional antiurolithic herb indicates the beneficial effect on preventing renal stone formation at middle and high dose. They show diuretic action, citrate increase, Ox and Ca decrease, and antioxidative effect. However, given their similar effects, MDs is superior to HDs. High dose Pp shows significantly anti-inflammatory and antioxidative effects, and relatively weak prophylaxis of CaOx stone formation. The combination therapy middle dose Ds and LiC indicates obvious synergistic action on urinary citrate, which can increase 415%,53% and 24.3% compared with normal, Ds, and LiC groups, respectively. In addition, there is a beneficial effect on preventing renal stone formation when urinary volume, pH value and citrate increase, Ox decrease.
方法80只成年雄性Wistar大鼠随机分入八组(正常、对照、低中高剂量金钱草和石韦组)。经一周适应环境,5%草酸铵饲料喂养实验组大鼠一周后,以不同浓度水煎剂灌胃(1ml／次,2次／天)。第21天置入代谢笼留取尿液／24h标本后,10%水合氯醛腹腔注射麻醉大鼠后,经下腹部血管采集血液,摘取肾脏标本。检测血、尿标本各项理化指标,观察尿结晶、肾结晶沉积和病理改变,并测定肾和尿液中骨桥蛋白表达及肾组织氧化应激指标。另取,90只成年雄性Wistar大鼠随机分入九组(正常、对照、低中高剂量金钱草组、碳酸锂组和低中高剂量金钱草联合碳酸锂组)。干预方法同上留取血、尿和肾组织标本。检测血、尿标本各项理化指标,观察尿结晶、肾结晶沉积和病理改变。
结果结石模型大鼠尿草酸水平升高3倍,尿钙水平下降5倍。中高剂量金钱草组尿量／24h、尿柠檬酸浓度明显升高,尿钙和草酸浓度下降(P<0.05)。在高剂量石韦组血钙和尿钙水平均有不同程度下降。正常大鼠肾组织几乎没有结晶沉积和病理改变,尿中亦无草酸钙结晶,亨利氏袢和肾小管上皮细胞表面OPN蛋白呈弱表达。结石大鼠肾组织多部位存在结晶沉积(以皮髓质交界处为主),肾小管显著扩张并结晶簇存在,并伴有大量炎细胞浸润；尿液中存在大量长柱形的一水草酸钙和菱形的二水草酸钙结晶。在中高剂量金钱草组,相对于结石模型大鼠而言,肾组织结晶沉积评分明显降低,而两组之间没有统计学差异；病理仅表现出小管轻度扩张和炎症浸润,OPN蛋白表达降低。尿晶体数量明显减少,分别为4.63±2.33、5.67±1.73和14.6±3.72(P<0.001)。高剂量石韦组呈现相似变化,但炎细胞浸润明显减少。结石大鼠NO和MDA水平迅速升高,SOD活性则被抑制(P<0.05)。尽管在高剂量金钱草和中剂量石韦组NO水平下降,SOD活性得到恢复,但MDA水平没有发生改变。在中剂量金钱草和高剂量石韦组上述指标均恢复接近正常值。仅在碳酸锂及联合治疗组检测到血清锂水平,且呈逐渐增高趋势,高剂量金钱草联合治疗组为1.38±0.41mmol／L。在碳酸锂和中高剂量联合治疗组,尿pH值、柠檬酸、钙水平明显升高。中高剂量联合治疗组的尿柠檬酸水平较中高剂量金钱草组增高53%,较碳酸锂组增高24.3%,尿量／24h增加1倍,尿草酸水平降低1倍；光镜下偶见结晶体的沉积,结晶评分明显降低(P<0.05)。除低剂量金钱草组外,各实验组病理评分均显著降低,其中以中剂量联合治疗组评分最低为0.78±0.67。中高剂量联合治疗组尿中晶体分别减少至1.11±1.05和1.38±1.06。
结论金钱草作为传统治疗泌尿系结石的植物药,具有利尿、增加尿柠檬酸、降低尿草酸、升高尿钙水平和抗氧化作用,在中高剂量显示出明显的抑制结石形成作用。由于两者效果相似,故认为中剂量效果更佳。高剂量石韦有抗炎,抗氧化作用及相对较弱抑制结石形成作用。中剂量金钱草联合碳酸锂组对尿柠檬酸浓度表现出显著的协同调节作用,使尿柠檬酸水平较正常大鼠升高415%,较中高剂量金钱草升高53%,较单剂量碳酸锂升高24.3%。此外,尿量增加、尿pH值升高、尿草酸水平降低均有利于抑制结-石形成。
Objective The pathogenesis of CaOx stone is a gradually multifaceted process including crystal nucleation, growth, aggregation and retention. The prevalence of urinary stone ranged from 1% to 20% (average 10%) in the world wide due to various factors, such as geography, climate, race and diet. Minimally invasive techniques were widely applied in clinic so that many patients with urolithiasis gained an effective management. However, both the high recurrence rate and complications are so far a serious problem and remains to be decreased. Even though thiazide diuretics and alkali-citrate have been proven to be effective in reducing calciuria and stone recurrence, undesirable side-effects limit their use in long-term medical treatment. Therefore, finding an effective alternative with few adverse-effects is becoming a new tendency. Currently, there has been a sustained concern on medicinal plants to prevent stone recurrence owing to their efficiency, economy, and safety. Recently, the new drugs, which based on medicinal plants, such as Paclitaxel, Ginkgo biloba, Camptothecin, Artemisinin, and so on, are used in clinic and cause a resurgence of interest in medicinal plants. In traditional chinese medicine (TCM), Desmodium styracifolium (Ds) and Pyrrosiae petiolosa (Pp) have been widely used to release renal colic, hematuria, and remove stones. Additionally, our previous studies has shown that Lithium carbonate can significantly modulate endogenous citrate level according to lithium inhibit sodium dicarboxylate co-transporters (NaDC), which can transport citrate from tubular into cells. Due to calculus is the result of multifactor, we propose to increase citrate regulation through the combination the multi-target effect of chinese herbs and single target effect of LiC, enhance the prophylaxis of CaOx stones, select the optimal formula and clear its possible mechanism.
Methods 80 adult male Wistar rats were randomly divided into eight groups (Normal, Control, LDs, MDs, HDs, LPp, MPp, and HPp). After one week of acclimatization, the rats were fed by containing 5% ammonium oxalate (AmOx) feed for one week, and then given the different does aqueous extracts(lml/time, Bid). On the 21st day of experimental period, the rats were placed in separate metabolic cages to collect 24-h urine, and then were anesthetized intraperitoneally with 10% Chloral Hydrate. Blood was obtained via inferior vena cava and then immediately excised kidneys. Samples were used to various determinations involving physical and biochemtry examination, pathological examination, crystalluria and CaOx deposition evaluation, OPN immunohistochemical staining, and oxidative stress studies.And then,90 adult male Wistar rats were randomly divided into eight groups (Normal, Placebo, LDs, MDs, HDs, LiC, LDs+LiC, MDs+LiC, HDs+LiC). The same interventions were 'used to treat the rats with the different doses aqueous extracts. The blood,24-h urine and kidneys were collected and used to various determinations involving physical and biochemtry examination, crystalluria, CaOx deposition evaluation and pathological examination.
Results There were 3-fold increase in urinary Ox level and 5-fold decrease in Ca excretion in the urolithic rats.24h urinary volume, Ca, and Cit level were increased with varying degrees in the MDs and HDs group, and Ox concentration decreased (P<0.05). Not only was serum Ca distinctly decreased, but. also urinary Ca reduced in HPp group. In the normal group, no CaOx deposition was detected without pathological injury and CaOx crystalluria. OPN protein expression was weak and observed in the loop of Henle and papillary surface epithelium of few cells. Urolithic rats were observed more crystals dispositions in all parts of the kidney, especially in cortico-medullary junction, severe dilation of tubules and massive inflammatory infiltration, and more columnar whewellite and pyramidal weddellite crystals existed in urine. Scoring of crystal deposition showed a significantly lower number of deposits in MDs and HDs groups, but no statistic difference was concluded between two groups. In MDs and HDs groups, scoring of crystal deposition showed a significantly lower number of deposits, but no statistic difference was concluded between two groups. Pathological alteration reduced and has slight dilation of tubules and inflammatory infiltration. Simultaneously, OPN showed significantly lower expression than those in the urolithiatic rats. The mean number of crystals was distinctly reduced (MDs, HDs vs. Control:4.63±2.33,5.67±1.73 vs.14.6±3.72, P<0.001). Similar results as well as slight inflammatory infiltration were found in high dose Pp group. NO and MDA level rose rapidly, but SOD activities were inhibited in the control group (P<0.05). Although lower NO and higher SOD level were significantly observed in HDs and MPp groups, there was no Statistical significance in MDA. However, in MDs and HPp groups above three indicators restored to near normal levels. Serum Li was only detected in Lic and combination therapy groups, and showed a gradually increasing trend. Moreover, serum Li level was 1.38±0.41mmol/L in high dose combination therapy group. Urinary pH value, citrate, and calcium concentration significantly increased. Of these, urinary citrate level increased 53% and 24.3% compared with Ds and LiC groups, respectively. There was 1-fold increase in urinary volume and 1-fold decrease in Ox excretion. Crystal depositions were few, and its score showed a significantly lower number of deposits in MDs+LiC and HDs+LiC groups(P<0.05). The score of experiment groups except for LDs group demonstrated distinct reduction, and the score of MDs+LiC is lowest (0.78±0.67). Urinary crystals reduced to 1.11±1.05 and 1.38±1.06.
Conclusions DS as a traditional antiurolithic herb indicates the beneficial effect on preventing renal stone formation at middle and high dose. They show diuretic action, citrate increase, Ox and Ca decrease, and antioxidative effect. However, given their similar effects, MDs is superior to HDs. High dose Pp shows significantly anti-inflammatory and antioxidative effects, and relatively weak prophylaxis of CaOx stone formation. The combination therapy middle dose Ds and LiC indicates obvious synergistic action on urinary citrate, which can increase 415%,53% and 24.3% compared with normal, Ds, and LiC groups, respectively. In addition, there is a beneficial effect on preventing renal stone formation when urinary volume, pH value and citrate increase, Ox decrease.
引文
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