【作者】 马晓红；

【导师】 王小琴；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的探讨左归丸对5/6肾大部切除模型并ROD大鼠甲状旁腺、骨、肾脏多脏腑、多靶点的影响,研究左归丸防治ROD的作用机制,探讨补肾中药替代骨化三醇治疗SHPT的可行性及理论依据,为临床治疗CKD并发ROD的中药新药研究提供靶点依据。方法SPF级Wistar大鼠分为6组：正常对照组、假手术组、左归丸组、中药方组、骨化三醇组、模型组(每组n=8)。分别药物干预4周、8周、12周,检测血Cr、BUN、Hb、ALP、iPTH、Ca、P;药物干预12周麻醉处死大鼠,双能X线测量大鼠股骨骨密度(BMD),检测PTHmRNA、CTmRNA、CYP27B1mRNA表达,检测PTH、CT、CYP27B1蛋白水平,观察肾脏组织形态学改变。结果血Cr,药物干预4周时,正常对照组明显低于各组(P<0.01),模型组明显高于各组(P<0.01)；药物干预8周时,除假手术组外,其余各组明显上升,且明显高于正常对照组(P<0.01),骨化三醇组、中药方组、假手术组明显低于模型组(P<0.01),中药方组明显低于左归丸组、骨化三醇组(P<0.01)(P<0.05)；药物干预12周时,除假手术组外,各组明显高于正常对照组(P<0.01),中药方组明显低于模型组、骨化三醇组、左归丸组(P<0.01)(P<0.05)。血BUN,药物干预4周时,除假手术组外,正常对照组明显低于各组(P<0.01),中药方组、假手术组明显低于模型组(P<0.05)(P<0.01)；药物干预8周时,正常对照组明显低于各组(P<0.01),中药方组明显低于左归丸组、骨化三醇组(P<0.05)；药物干预12周时,各组明显高于正常对照组(P<0.01) (P<0.05),中药方组、骨化三醇组明显低于模型组(P<0.01) (P<0.05)。Hb,药物干预4周时,正常对照组明显高于各组(P<0.01),骨化三醇组、假手术组明显高于模型组(P<0.01)；药物干预8周时,正常对照组明显高于各组(P<0.01) (P<0.05),中药方组明显高于左归丸组、骨化三醇组和模型组(P<0.01)(P<0.05)(P<0.01)；药物干预12周时,各组明显低于正常对照组(P<0.01)(P<0.05),中药方组明显高于模型组(P<0.01)。血清ALP,在药物干预4周时,骨化三醇组明显低于正常对照组(P<0.05),左归丸组、骨化三醇组明显低于中药方组(P<0.01)；药物干预8周时,模型组明显低于正常对照组(P<0.01)、高于假手术组(P<0.05)；药物干预12周时,模型组明显高于正常对照组、骨化三醇组、假手术组(P<0.05) (P<0.01),左归丸组明显高于骨化三醇组(P<0.01)。iPTH,药物干预4周时,模型组、左归丸组明显高于正常对照组、假手术组(P<0.01) (P<0.05),中药方组、骨化三醇明显低于模型组(P<0.05),中药方组明显低于左归丸组(P<0.01)；药物干预8周时,模型组明显高于中药方组、左归丸组、骨化三醇组(P<0.01)；骨化三醇组明显高于左归丸组、中药方组(P<0.01)(P<0.05)；药物干预12周时,正常对照组明显低于各组(P<0.01),模型组明显高于各组(P<0.01)。血清Ca,药物干预4周时,中药方组、假手术组和左归丸组明显高于模型组(P<0.01)和(P<0.05)；药物干预8周时,模型组明显低于假手术组(P<0.01),左归丸组明显低于模型组、骨化三醇组(P<0.05)(P<0.01)；药物干预12周时,模型组明显高于各组(P<0.01)(P<0.05)。血清P,药物干预4周时,模型组明显高于各组(P<0.01)；药物干预8周时,模型组明显高于各组(P<0.01),左归丸、中药方组明显高于骨化三醇组(P<0.01)(P<0.05)；药物干预12周时,模型组明显高于各组(P<0.01)(P<0.05),中药方组明显低于左归丸组(P<0.01)。BMD,模型组明显低于各组(P<0.01)(P<0.05),中药方组明显低于左归丸组、骨化三醇组(P<0.01)。PTHmRNA,正常对照组与假手术组、中药方组与模型组没有显著性差异(P>0.05),左归丸组明显低于骨化三醇组、中药组、模型组(P<0.01),骨化三醇组明显低于中药组、模型组(P<0.01)。CTmRNA正常对照组与假手术组、模型组没有显著性差异(P>0.05),左归丸组明显高于各组(P<0.01),中药组明显高于除左归丸以外的各组(P<0.01),骨化三醇组明显低于模型组(P<0.01)。PTH,各组蛋白表达明显高于正常对照组(P<0.01)；中药方组明显低于模型组、左归丸组、骨化三醇组(P<0.01)；左归丸组明显低于模型组、骨化三醇组(P<0.05)。CT,正常对照组的表达明显高于各组(P<0.01)；模型组明显低于中药方组、骨化三醇组及左归丸组(P<0.01)；左归丸组明显低于中药方组、骨化三醇组(P<0.01)。蛋白质甲状旁腺PCNA,在正常对照组中表达最弱,在模型组中表达最强,且两组有显著性差异(P<0.01)；骨化三醇组比中药方组表达弱(P<0.01),左归丸组比中药方组、骨化三醇组表达弱(P<0.01) (P<0.05)。CYP27B1mRNA,假手术组明显高于各组(P<0.01),模型组明显低于各组(P<0.01),左归丸组明显低于中药方组、骨化三醇组、模型组(P<0.01),中药方组与骨化三醇组没有显著性差异(P>0.05)。CYP27B1蛋白表达,模型组明显低于各组(P<0.01),左归丸明显低于中药方组、骨化三醇组(P<0.01),中药方组与骨化三醇组没有差异(P>0.05)。CYP27B1免疫荧光结果：正常对照组、假手术组表达强；模型组比各组表达弱(P<0.05)；中药方组较左归丸组、骨化三醇组表达强(P<0.01)；左归丸组比骨化三醇组表达弱(P<0.05)。结论5/6肾大部切除大鼠模型造模成功并发ROD表现。ROD药物的干预应从肾功不良初期进行,建立预防模式。左归丸能改善ROD的骨营养不良,对PTH有直接的抑制作用,其机制可能是通过调节钙磷代谢及参与成骨细胞代谢的途径。左归丸对甲状旁腺、甲状腺、骨有多重作用,可抑制甲状旁腺的增生,抑制破骨细胞活性,其对ALP的调节作用将预示在改善骨营养不良方面将比骨化三醇更有优势。左归丸和骨化三醇不能有效地控制Cr的升高以及Hb的降低,他们不能有效改善肾功能不良、纠正贫血。在血Cr、BUN、Hb水平控制方面,中药方组显示出的明显优势提示其可能与左归丸有互补效应。中药方干预能保护肾脏,左归丸与中药方合用可能在抑制SHPT、改善钙磷代谢、调动成骨系统、纠正贫血、延缓肾脏病的进展方面的效果会优于骨化三醇,两方可能有互补和协同效应。基于左归丸对其靶器官甲状旁腺、甲状腺、骨的影响,可能会成为临床替代骨化三醇对ROD进行早期干预的经方,佐以补肾生精益气活血中药,对慢性肾脏病进展的控制可能会有极大帮助。更多还原

【Abstract】 Objective To Discussion the influence of Zuoguiwan on the parathyroid, bone, and kidney of rat model with renal osteodystrophy (ROD) created by 5/6 renal resection. Moreover,the mechanisms were also investigated in order to provide more target proof in treating CKD complicating ROD by herbs of invigorating Shen.Methods Wistar rats of SPF grade were divided into six groups:normal control group, sham operation group, Zuoguiwan group, herbal group, Calcitriol group and model group (each n=8). Respective drug was administrated for 4 weeks,8 weeks,12 weeks in each group. Thereafter, the blood Cr, BUN, Hb,ALP, iPTH, Ca, P were tested;The rats were anesthetized and killed after drug 12-week intervention.The femur bone mineral density (BMD) was measured by dual energy X-ray. The expressions of PTHmRNA,CTmRNA,CYP27B1mRNA were detected by Real-time PCR and the protein levels of PTH,CT,CYP27B1 were also determined by western blotting. In addition,the morphologic changes of kidney was observed.Results Serum Cr levels of normal control group was significantly lower than other groups after 4-week intervention(P<0.01). In the model group,the levels of Cr was higher than that of other groups (P<0.01). Eight weeks after intervention,the serum Cr levels of other groups had significantly increased except to sham group,the Cr level of the other group was significantly higher than the control group (P<0.01). In the Calcitriol groud,herbal group and sham operation,the levels of serum Cr were significantly lower than that of the model group (P<0.01).Indeed,level of Cr the herbal group much significantly lower when compared with the Zuoguiwan group and Calcitriol group(P<0.01 or P<0.05).Excepting to the sham group,the Cr level of the other groups were significantly higher than normal control group 12 weeks after drug intervention (P<0.01).In the herbal group,the Cr level of was significantly lower than that of the model group,Calcitriol group and Zuoguiwan group(P<0.01 or P<0.05).Serum BUN levels of the normal group was significantly lower than other group after 4-week intervention excepting to the sham group(P <0.01).In herbal group and sham operation group,the levels of BUN were significantly lower than that of model group(P<0.05 or P<0.01).Eight weeks after intervention,the BUN level of normal control group was significantly lower than other group (P<0.01).In the herbal group,the level of BUN was significantly lower than that of Zuoguiwan group and calcitriol group(P<0.05).The BUN level of the other group was significantly higher than the control group 12 weeks after drug intervention(P<0.01 or P<0.05).In the herbal group and Calcitriol group,the BUN level of were significantly lower than that of model group(P<0.01 or P<0.05).Serum Hb levels of the normal control group was significantly higher than other group after 4-week intervention(P<0.01).In the Calcitriol group and shan operation group,the level of Hb were higher than that of model group(P<0.01).Eight weeks after intervention,the Hb level of normal control group was significantly higher than other group(P<0.01 or P<0.05).In the herbal group,the level of Hb was significantly higher than that of Zuoguiwan group,Calcitriol group and model group(P<0.01orP<0.05or P<0.01).The Hb level of the other group was significantly lower than that of the control group 12 weeks after drug intervention(P<0.01 or <0.05). The Hb level of herbal group was significantly higher than that of model group (P<0.01).Serum ALP level in Calcitriol group was significantly lower than that in normal control group (P<0.05) after 4-week intervention. The ALP levels in Zuoguiwan group and Calcitriol group were markedly lower than that in herbal group (P<0.01).Eight weeks after intervention, the ALP level of model group was significantly lower than that of the control group (P<0.01) and was significantly higher sham operation group(P<0.05).The ALP level of the model group was significantly higher than that of the control group, Calcitriol group and sham operation group respectively 12 weeks after drug intervention(P<0.05 or P<0.01). The ALP level of Zuoguiwan group was significantly higher than that of Calcitriol group (P<0.01).Serum iPTH levels of model group and Zuoguiwan group were significantly higher than those of normal control group and sham operation group after 4-week intervention(P<0.01 or P<0.05). In the herbal group and Calcitriol group,the levels of iPTH were lower than that of the model group (P<0.05). Compared with the Zuoguiwan group,the herbal group had significantly lower level of iPTH(P<0.01).Eight weeks after intervention,the iPTH level of the model group was significantly higher than that of herbal group,Zuoguiwan group and Calcitriol group(P<0.01). In addition,Zuoguiwan group and herbal group had lower levels of iPTH than Calcitriol group (P <0.05 or P＜0.01).12 weeks after drug intervention,the iPTH level of the normal control group was significantly lower than other groups (P<0.01),the iPTH level of model group was significantly higher than other groups (P <0.01).Serum Ca levels of herbal group,sham operation group and Zuoguiwan group were significantly higher than model group 4 weeks after drug intervention (P<0.01 or P<0.05).Eight weeks after intervention,the Serum Ca level of the model group was significantly lower than sham operation group (P <0.01),In the Zuoguiwan group,the levels of Ca were lower than that of the model group and Calcitriol group (P<0.05 or P<0.01).Serum Ca levels of the model group was significantly higher than other groups 12 weeks after drug intervention (P<0.01 or P<0.05).Serum P levels of model group was significantly higher than other groups after 4-week intervention(P<0.01). Eight weeks after intervention,the Serum P level of model group was significantly higher than other groups(P<0.01),In the Zuoguiwan group and herbal group,the Serum P level of were higher than that of calcitriol group(P<0.01 or P<0.05). The P level of the model group was significantly higher than other groups 12 weeks after drug intervention(P<0.01 or<0.05). The P level of herbal group was significantly lower than Zuoguiwan group(P<0.01).The BMD of the model group was significantly lower than other groups(P<0.01 or P<0.05).In the herbal group,the BMD was significantly lower than that of Zuoguiwan group and Calcitiol group (P<0 .01).PTHmRNA levels of betweening control group and sham operation group,herbal group and model group had not significant difference(P>0.05).In the Zuoguiwan group,the PTHmRNA level of was significantly lower than that of Calcitriol group,herbal group and model group(P<0.01). Indeed,level of PTHmRNA the Calcitriol group much significantly lower when compared with the herbal group and model group(P<0.01).CTmRNA levels of betweening control group and sham operation group,herbal group and model group had not significant difference(P > 0.05).In the Zuoguiwan group,the CTmRNA level of was significantly higher than that of other group(P<0.01). Excepting to Zuoguiwan group,compared with the herbal group,other group had significantly higher level of CTmRNA(P<0.01). CTmRNA levels of Calcitriol group was significantly lower than that of model group(P<0.01).The PTH protein expression of the other group was significantly higher than that of the control group(P<0.01).In the herbal group,the PTH of was significantly lower than that of model group,Zuoguiwan group and Calcitriol group(P<0.01). In addition,Zuoguiwan group and model group,Calcitriol group had lower protein expression of PTH(P<0.05).The CT protein expression of the control group was significantly higher than that of the other group(P<0.01).In the model group,the CT of was significantly lower than that of herbal group,Calcitriol group and Zuoguiwan group(P<0.01).In addition,Zuoguiwan group and herbal group,Calcitriol group had lower protein expression of CT(P<0.01).The PCNA expression of the normal control group was the weakest, in the model group,was the strongest expression and had significantly differ(P<0.01).Compared with the Calcitriol group,the herbal group had significantly weaker expression of PCNA(P<0.01).In the Zuoguiwan group, the expression of PCNA was weaker than that of the herbal group and Calcitriol group(P<0.01 or P<0.05).CYP27B1mRNA levels of sham operation group was significantly higher than the other group(P<0.01). CYP27B1mRNA levels of the model group was significantly lower than that of other group(P<0.01).In the Zuoguiwan group,the CYP27B1mRNA level of was significantly lower than that of herbal group,Calcitriol group and model group(P<0.01). Compared with the herbal group,Calcitriol group had not significantly differ level of CYP27B1mRNA (P> 0.05).The CYP27B1 protein expression of the model group was significantly lower than the other group(P<0.01).In Zuoguiwan group,the CYP27B1 of was significantly lower than that of herbal group, Calcitriol group(P<0.01). Compared with the herbal group,Calcitriol group had not significantly differ the protein expression of CYP27B1 (P> 0.05).In the normal control group and sham operation group,the protein fluorescence expression of CYP27B1 were the strongest. Compared with the’ model group,the other group had significantly weaker fluorescence expression of CYP27B1(P< 0.05).CYP27B1 immunofluorescence expression of herbal group was significantly stronger than that of Zuoguiwan group and Calcitriol group(P< 0.01).Compared with the Zuoguiwan group,calcitriol group had signifiticantly weaker the protein fluorescence expression of CYP27B1(P< 0.05).Conclusion ROD rat model of 5/6 renal resection are successfully created.Treatment or prevention with traditional Chinese herb or Zuoguiwan should began from incipience of renal dysfunction.Zuoguiwan can improve renal osteodystrophy bone malnutrition, and has a direct inhibitory effect on parathyroid hormone,which might be contributed to the regulation of calcium,phosphorus and bone cell metabolism.Zuoguiwan may play a role on glandulae parathyroideae, glandula thyreoideaand bone may inhibit parathyroid hyperplasy and osteoclast cytoactive.The regulation on the ALP suggest that Zuoguiwan is better than Calcitriol in improving osteodystrophy.Zuoguiwan and Calcitriol can not effectively control the Cr,increase Hb, improve renal dysfunction and anemia. However,serum Cr,BUN,Hb level could be improved by the treatment in the herbal group which suggested that they are complementory. TCM treatmen can protect kidney function.The two groups combined with each other may be better than Calcitriol treatment in inhibiting secondary hyperparathyroidism,regulating calcium and phosphorus metabolism,mobilizing bone formation system,correcting anaemia and defering renal disease. Two treatments can be complementory and synergistic.Base on the effect of Zuoguiwan on target organ glandula thyreoidea, glandulae parathyroideae and bone,Zuoguiwan should became a classical prescriptions that ROD is treated early to instead of Calcitriol in clinic,adding to herbs of invigorating Shen to generate marrow and supplementing qi and activating blood circulation,they could had actively help for control of disease advancement of chronicity renal.更多还原